CHARACTERIZATION OF PRE-CANCER BIOMARKER FOR PROGNOSTIC SCREEN

Abstract

The invention features compositions and methods for a pre-cancer prognostic screen.

Claims

1. A method of detecting protein arginine methyltransferase 8 (PRMT8) variant 2 in a sample comprising: obtaining a test sample from a subject; contacting the test sample with a detectable probe capable of binding PRMT8 variant 2 nucleic acid or protein, or fragment thereof, and detecting binding between the detectable probe and the PRMT8 variant 2 nucleic acid or protein.

2. The method of claim 1, wherein the subject is a human.

3. The method of claim 1, further comprising administering a chemotherapeutic agent, radiation therapy, cryotherapy, or hormone therapy, thereby inhibiting tumor cell growth in said subject.

4. The method of claim 3, wherein the chemotherapeutic agent comprises doceaxel, cabazitaxel, mitoxantrone, estramustine, doxorubicin, etoposide, or paclitaxel.

5. The method of claim 1, further comprising administering an anti-neoplastic agent, wherein said anti-neoplastic agent comprises radiotherapy, a cell death-inducing agent, or a proteasome inhibitor, thereby inhibiting tumor cell growth in said subject.

6. The method of claim 1, wherein said test sample comprises ribonucleic acid (RNA).

7. The method of claim 6, wherein the expression level of PRMT8 messenger ribonucleic acid (mRNA) is determined.

8. The method of claim 7, wherein reverse transcription polymerase chain reaction (RT-PCR) is utilized to determine a level of PRMT8 variant 2 mRNA in said sample.

9. The method of claim 1, wherein said PRMT8 in said test sample comprises a PRMT8 variant 2 mRNA variant comprising the nucleic acid sequence set forth in SEQ ID NO: 8.

10. The method of claim 1, further comprising administering an inhibitor of PRMT8 to said subject, thereby inhibiting tumor cell growth.

11. The method of claim 10, wherein said inhibitor of PRMT8 comprises a small molecule inhibitor, RNA interference (RNAi), an antibody, or any combination thereof.

12. The method of claim 1, further comprising comparing the expression level of PRMT8 variant 2 in said test sample with the expression level of PRMT8 variant 2 in a reference sample, wherein the reference sample comprises a tissue-matched normal control sample.

13. The method of claim 1, wherein said test sample comprises a plasma sample, a blood sample, or a tissue sample.

14. The method of claim 1, further comprising comparing the expression level of PRMT8 variant 2 in said test sample with the expression level of PRMT8 variant 2 in a reference sample, wherein the reference sample is obtained from a healthy normal control subject.

15. An isolated PRMT8 polypeptide variant.

16. The isolated PRMT8 polypeptide variant of claim 15, wherein said isolated PRMT8 polypeptide variant comprises a synthetic isolated PRMT8 polypeptide variant.

17. The isolated PRMT8 polypeptide variant of claim 15, wherein said polypeptide variant comprises an amino acid sequence set forth in SEQ ID NO: 7.

18. An isolated nucleotide sequence encoding the isolated PRMT8 polypeptide variant of claim 15.

19. The isolated nucleotide sequence of claim 18, wherein said isolated nucleic acid sequence comprises a synthetic isolated nucleic acid sequence.

20. The isolated nucleic acid sequence of claim 19, wherein said isolated nucleic acid sequence comprises complementary deoxyribonucleic acid (cDNA).

21. The isolated nucleotide sequence of claim 20, wherein said isolated nucleic acid sequence is immobilized on a solid support.

22. The isolated nucleic acid sequence of claim 21, wherein said isolated nucleic acid sequence is linked to a detectable label.

23. The isolated nucleic acid sequence of claim 22, wherein said detectable label comprises a fluorescent label, a luminescent label, a chemiluminescent label, a radiolabel, a SYBR Green label, or a Cy3-label.

24. A kit for detecting the expression of PRMT8 mRNA comprising a PRMT8-specific primer.

25. The kit of claim 24, wherein the PRMT8-specific primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 5, SEQ ID NO: 2 and SEQ ID NO: 6, SEQ ID NO: 3 and SEQ ID NO: 6, and SEQ ID NO: 4 and SEQ ID NO: 6.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0072] FIG. 1 is a schematic of protein variants of PRMT8 and their localization.

[0073] FIG. 2A is a bar chart showing the five most up- and down-regulated chromatin modifiers in extended lifespan (ELS) cells compared to control cells.

[0074] FIG. 2B is a photograph of a blot showing the results of RT-PCR of PRMT8 in various cell types compared to actin.

[0075] FIG. 2C is a photograph of a western blot of PRMT8 in various cell types compared to actin.

[0076] FIG. 3 is a series of photomicrographs showing PRMT8 localization in ELS and control (Ctl) cells.

[0077] FIG. 4 is a photograph of a blot showing PRMT8 variant transcript expression by RT-PCR in ELS cells and hESCs.

[0078] FIG. 5A is a line graph showing cumulative population doublings of control cells under normal conditions, with a scramble negative control, and with three separate PRMT8 short hairpin ribonucleic acids (shRNAs).

[0079] FIG. 5B is a series of photomicrographs showing immunocytochemistry of cells from each treatment group at passage 10.

[0080] FIG. 5C is a series of photomicrographs showing immunocytochemistry of cells from each treatment group at passage 13, 15 days after being transferred to ELS culture conditions.

[0081] FIG. 6 is a schematic showing derivation of the induced regeneration competent (iRC) phenotype. Supplementation with the growth factor FGF2 under reduced oxygen over 7 day culture period leads to increased plasticity of adult human fibroblasts characterized by a proregenerative, non-tumorigenic phenotype.

[0082] FIG. 7A-FIG. 7B is a series of bar charts showing the effect of culture conditions on expression of chromatin modification enzymes. Target genes were analyzed using a qRT-PCR Chromatin Modification Enzyme Array (SA Biosciences) (n=1). FIG. 7A shows fold change in expression of the top 5 most up- and down-regulated chromatin modifiers as normalized to the housekeeping gene RPL13A. FIG. 7B shows fold change in expression in members of the PRMT family normalized to the housekeeping gene RPL13A.

[0083] FIG. 8A-FIG. 8D is a series of photographs of blots and a bar chart showing the effect of culture conditions on PRMT8 expression. FIG. 8A shows PRMT8 transcript expression in control human dermal fibroblasts (CRL-2352), iRC cells, and hESCs compared to expression in mouse brain by RT-PCR. Actin was used as a loading control. FIG. 8B shows PRMT8 protein expression in control human dermal fibroblasts (CRL-2352), iRC cells, and hESCs compared to expression of purified GST-tagged PRMT8. Actin was used as a loading control. 10 g of total protein were loaded in each lane except for GST-PRMT8 lanes (0.25 g and 0.5 g, respectively). Antibody dilutions are as follows: PRMT8-1:200, actin-1:5000, HRP anti-Rb-1:10,000. FIG. 8C shows densitometric representation of protein levels normalized to actin from three separate experiments. *All treatments were significantly different from each other: control compared to iRC, p=0.0002; control compared to hESCs, p=0.002; iRC compared to hESCs, p=0.02. FIG. 8D shows PRMT8 transcript expression in various control human dermal fibroblast lines (CRL-2352; CRL-2097; CT-1005) compared to the same lines grown under iRC conditions by RT-PCR. Actin was used as a loading control.

[0084] FIG. 9A and FIG. 9B is a series of graphs showing PRMT8 transcript sequence. FIG. 9A shows a graphic representation of PRMT8 and amplicon location. Boxes represent the 10 exons of PRMT8 where the dashed line in exon 1 represents the lengths of the alternative 5 exons that differentiate variant 1 from variant 2. The grey line that spans a portion of exons 8-10 represents the amplicon that was sequenced. FIG. 9B shows PRMT8 cDNA from iRC cells was cloned intopLVX at Smal (CCCGGG), and sequenced. The grey line in the sequencing data represents the grey amplicon in FIG. 9A.

[0085] FIG. 10A and FIG. 10B are a series of schematics showing graphic representation of PRMT8 transcript variants, protein isoforms, and localization differences. FIG. 10A shows the genomic alignment of PRMT8 transcript variants 1 and 2. Numbers represent million base pairs, dashed lines represent introns, and solid vertical lines represent exons. FIG. 10B shows both PRMT8 mRNA variants are expressed from the PMRT8 gene on chromosome 12. mRNA variant 1 has 3 alternative translation start sites, responsible for protein isoforms 1-3. mRNA variant 2 is transcribed from an alternative 5 exon and is responsible for translation of isoform 4. Isoform 1 harbors an N-terminal myristoylation motif, represented by the red coil, conferring plasma membrane localization. Isoforms 2 and 3 are truncated at the N-terminus and display nuclear localization. Isoform 4 is a variant that has not been explored experimentally. Conserved PRMT core regions are represented in grey, methyltransferase domains are represented in black, and the conserved THW loop is represented in blue. The unique portion of the protein sequence for isoform 4 is represented in orange.

[0086] FIG. 11A-FIG. 11D are a series of graphs and a photograph of a blot showing PRMT8 variant expression. FIG. 11A shows the sequence for 5 RACE of hESCs-PRMT8 compared to sequences in the NCBI database. Asterisks represent base pairs mismatched from the NCBI database. Dashed line represents beginning of alignment with database sequences, which begins with the 172nd nucleotide. Solid lines represent exon-exon junctions. FIG. 11B shows the sequence for 5 RACE of iRC-PRMT8 compared to sequences in the NCBI database. Asterisks represent base pairs mismatched from the NCBI database, and N represents sequencing misreads. Solid lines represent exon-exon junctions. FIG. 11C shows graphic representation of 5 RACE data from hESCs and iRC cells compared to variant 1 and variant 2. Solid horizontal lines represent mRNA alignments between treatments and boxes represent exons. Numbers delineate base pairs. FIG. 11D shows variant specific PRMT8 transcript expression in control cells, iRC cells, and hESCs using RT-PCR. Actin was used as a loading control.

[0087] FIG. 12A and FIG. 12B are a series of photomicrographs and a photograph of a blot showing demonstration of knockdown specificity. FIG. 12A shows GFP reporter fluorescence of U87MG glioblastomas on day 2 post-transduction. Scale bars are 200 m. FIG. 12B shows transcript expression for PRMT8 variant 2 is compared to PRMT1 in all treatment groups using RT-PCR. Actin and GFP were used as loading controls. Cells were harvested 2 days post-transduction.

[0088] FIG. 13A and FIG. 13B is a line graph and a series of photomicrographs demonstrating the effect of PRMT8 on human dermal fibroblast growth and longevity. FIG. 13A shows three independent replicates were performed and cumulative population doublings were measured and averaged for cells in all treatment groups. Error bars represent standard deviation. FIG. 13B shows GFP reporter fluorescence on day 6 (left) and day 14 (right).

[0089] FIG. 14A and FIG. 14B is a line graph and a series of hotomicrographs demonstrating the effect of PRMT8 on glioblastoma growth and longevity. FIG. 14A shows that three independent replicates were performed and cumulative population doublings were measured and averaged for cells in all treatment groups. Error bars represent standard deviation. Measurements for cells in the PRMT8 shRNA treatment group were terminated after day 6 due to complete cell loss. FIG. 14B shows GFP reporter fluorescence on day 1 (left) and day 6 (right).

DETAILED DESCRIPTION

[0090] The invention is based, at least in part, on the surprising discovery that an mRNA variant of PRMT8 is upregulated in cells that resemble pre-cancer. As described in detail below, PRMT8 is used as a biomarker in a simple, inexpensive test to identify pre-cancerous cells. Specifically, as described herein, an mRNA variant of PRMT8 was identified in cells with a prolonged life span and acquired regeneration potential, but without the ability to form tumors. The marker appears before the cells become turmorigenic and can be used for detection of a pre-cancerous state.

[0091] Cancer is one of the most prevalent diseases worldwide, accounting for 25% of all deaths in the United States (Siege et al., 2012 Cancer statistics, 2:10-29). As such, medicine has shifted from reactive to proactive therapies. Colonoscopies alone have reduced morality from colorectal cancer by 53% (Zauber et al., 2012 New England Journal of Medicine, 366:687-696). As medical technology advances, preventative screenings are becoming less invasive and more widespread as research reveals biomarkers that can be used to identify cancer-related changes. However, prior to the invention described herein, there were no biomarkers widely used in cancer screens prior to tumor formation. As such, described herein is a prognostic test that intervenes before patients develop cancer by screening for biomarkers of pre-cancerous biological changes with a qualitative diagnostic screening device that detects a biomarker associated with pre-cancerous cells.

[0092] Finite cellular proliferative lifespan and onset of irreversible growth arrest, termed senescence, has long been recognized in differentiated eukaryotic cells (Kyo et al., 2008 Cancer Science, 99: 1528-1538). Molecular mechanisms that regulate this terminal arrest of the cell cycle, however, can be deregulated, leading to uncontrolled cell proliferation in cancer cells or continuous self-renewal in pluripotent stem cells; both cell types becoming neoplastic in parallel. Six biological capabilities have been detailed during the evolution of healthy cells to a neoplastic state. Of the 6 canonical hallmarks of cancer (resisting cell death, sustaining proliferative signaling, evading growth suppressors, activating invasion and metastasis, enabling replicative immortality, and inducing angiogenesis), four are associated with increased cellular lifespan (Hanahan, D. and Weinberg, R. A. 2000 Cell, 100: 57-70; Hanahan, D. and Weinberg, R. A. 2011 Cell, 144: 646-674). Investigating processes that control lifespan enables progression toward identification of mechanisms that control the switch between normal cell division and neoplastic proliferation.

[0093] Methylation is one of the most widely studied and diverse post-translational modifications (PTMs). Methyl groups can be added to the side chains of various amino acids, such as proline, lysine, histidine, and arginine (Lee et al., 2005 Endocrine reviews, 26: 147-170). In particular, arginine methylation can influence biological processes such as transcriptional permissiveness, cellular differentiation, and telomere length and stability (Lee et al., 2005 Endocrine reviews, 26: 147-170; Wang et al., 2001 Science Signaling, 293: 853; Peterson, C. L. and Laniel, M. A. 2004 Current Biology, 14: R546-R551; Yu et al., 2006 Genes & Development, 20: 3249-3254; Iberg et al., 2008 Journal of Biological Chemistry, 283: 3006-3010; Mitchell et al., 2009 Molecular and Cellular Biology, 29: 4918-4934; Tee et al., 2010 Genes & Development, 24: 2772-2777). Many biological processes regulated by arginine methylation are well-described, but prior to the invention described herein limited knowledge existed about how PRMTs themselves are regulated. However, aberrant expression of protein arginine methyltransferase (PRMT) family members has been associated with cardiovascular and pulmonary diseases, as well as various types of cancers including lung, bladder, colon, and breast cancers (Yoshimatsu et al., 2011 International Journal of Cancer, 128: 562-573; Zakrzewicz et al., International Journal of Molecular Sciences, 13: 12383-12400; Mathioudaki et al., 2008 British Journal of Cancer, 99: 2094-2099; Goulet et al., 2007 Journal of Biological Chemistry, 282: 33009-33021).

Protein Arginine Methyltransferase 8 (PRMT8)

[0094] Arginine methyltransferases have remained grossly understudied given their critical functional roles and variant-specific functions in cancer biology. As described herein, evaluation of PRMT variant expression and regulation reveals critical physiological and pathophysiological mechanisms and leads to therapeutic developments.

[0095] Prior to the invention described herein, the function of the protein arginine methyltransferase 8 enzyme was largely uncertain. PRMT8 is a protein that is encoded by the PRMT8 gene in humans. Arginine methylation is a widespread posttranslational modification mediated by arginine methyltransferases, such as PRMT8. Arginine methylation is involved in a number of cellular processes, including DNA repair, RNA transcription, signal transduction, and protein compartmentalization. PRMT8 is a membrane-associated arginine methyltransferase that can both catalyze the formation of omega-N monomethylarginine (MMA) and asymmetrical dimethylarginine (aDMA). For example, PRMT8 binds and dimethylates Ewing sarcoma breakpoint region 1 (EWS) protein. A variety of biological roles for PRMT family members are being uncovered indicating potential regulatory mechanisms for arginine methylation in cellular senescence.

[0096] PRMT8 was first identified because of sequence similarity with PRMT1 (Lee et al., 2005 Journal of Biological Chemistry, 280: 32890-32896, incorporated herein by reference), and phylogenetic analysis revealed it to be a paralogue of PRMT1 in vertebrates (Hung, C. M. and Li, C. 2004 Gene, 340: 179-187; Lin et al., 2013 PLOS ONE, 8: e55221). PRMT1 is ubiquitously expressed and is found in both nuclei and cytoplasm (Sayegh et al., 2007 Journal of Biological Chemistry, 282: 36444-36453; Frankel et al., Journal of Biological Chemistry, 277: 3537-3543; Herrmann et al., 2005 Journal of Biological Chemistry, 280: 38005-38010). Although members of the PRMT family are all highly homologous, PRMT8 and PRMT1 are most similar with 83% sequence identity, differing only at the N-terminus, where PRMT8 contains 76 additional amino acids (Lee et al., 2005 Journal of Biological Chemistry, 280: 32890-32896; Sayegh et al., 2007 Journal of Biological Chemistry, 282: 36444-36453; Kousaka et al., 2009 Neuroscience, 163: 1146-1157). Northern blot analysis demonstrated that full-length PRMT8 transcript expression was found largely in brain tissue (Lee et al., 2005 Journal of Biological Chemistry, 280: 32890-32896; Sayegh et al., 2007 Journal of Biological Chemistry, 282: 36444-36453; Taneda et al., 2007 Brain Research, 1155: 1-9). However, analysis of PRMT8 in a non-mammalian vertebrate system found ubiquitous expression during embryonic development, whereas expression only became restricted to brain tissue after neural development (Lin et al., 2013 PLOS ONE, 8: e55221).

[0097] PRMT8 has three described isoforms with unique N-termini translated from differing inframe start codons. Early characterization of full length PRMT8 (isoform 1) revealed a glycine residue at the N-terminus modified by a myristoylation motif (Lee et al., 2005 Journal of Biological Chemistry, 280: 32890-32896; Sayegh et al., 2007 Journal of Biological Chemistry, 282: 36444-36453, each of which is incorporated herein by reference). Myristoylation is the addition of a hydrophobic moiety that results in sequestration of modified proteins to the plasma membrane (Lee et al., 2005 Journal of Biological Chemistry, 280: 32890-32896; Sayegh et al., 2007 Journal of Biological Chemistry, 282: 36444-36453). However, overexpressed PRMT8 translated from the second (isoform 2) and third (isoform 3) in-frame start codons displays nuclear localization (Kousaka et al., 2009 Neuroscience, 163: 1146-1157, incorporated herein by reference). In mice, endogenous PRMT8 localizes to nuclei (Kousaka et al., 2009 Neuroscience, 163: 1146-1157). Previous studies of PRMT8 utilized overexpression of the full-length isoform, which guided the consensus that the endogenous isoform is the full-length product and that expression is restricted to brain tissue. If PRMT8 is one of the truncated nuclear isoforms and is expressed more widely than initially reported, it challenges the existing paradigm and it suggests that PRMT8, like other PRMT family members, may have a role in critical cellular processes through chromatin modification or regulation of protein-protein interactions. However, prior to the invention described herein, the expression or function of PRMT8 in human cells was not examined.

[0098] An exemplary human PRMT8 amino acid sequence (PRMT8 isoform 1) is set forth below (SEQ ID NO: 20; GenBank Accession No. NP_062828, Version NP_062828.3 (GI:74099699), incorporated herein by reference):

TABLE-US-00004 1mgmkhssrclllrrkmaenaaestevnsppsqppqpvvpakpvqcvhhvstqpscpgrgk 61mskllnpeemtsrdyyfdsyahfgiheemlkdevrtltyrnsmyhnkhvfkdkvvldvgs 121gtgilsmfaakagakkvgfiecssisdysekiikanhldniitifkgkveevelpvekvd 181iiisewmgyclfyesmlntvifardkwlkpgglmfpdraalyvvaiedrqykdfkihwwe 241nvygfdmtcirdvamkeplvdivdpkqvvtnaclikevdiytvkteelsftsafclqiqr 301ndyvhalvtyfnieftkchkkmgfstapdapythwkqtvfyledyltvrrgeeiygtism 361kpnaknvrdldftvdldfkgqlcetsvsndykmr

[0099] An exemplary human PRMT8 nucleic acid sequence (PRMT8, transcript variant 1, mRNA) is set forth below (SEQ ID NO: 21; GenBank Accession No. NM_019854, Version NM_019854.4 (GI:374858038), incorporated herein by reference):

TABLE-US-00005 1gtgttgcttcgcccagcggatcggcagaagttgagaggagttggcggctgcctccggccg 61gccggactttgcgagcagcctggagaggatccgcgaccgccgccgccgccgccgcggagg 121cttcggggctgcttccctcgagcttagcccgcagcgcgggtggagaggggcggggagggg 181gtcgggggcacgagaagaacttgaaaccgtgtgaaggaatccggagcagatgagaaggga 241ggaaaataaaagaaagtggagactgcagaacagactccgctgtggctgactgtgccggcc 301gacgctccagctgaggggctgggttggatttttttttttctcccatcctctcgctctctc 361ttttaaagcgacaccagctctctctcctcctctactatctcggtatcaccaaacccttgc 421cggctcttatgggcatgaaacactcctcccgctgcctgctcctgaggaggaaaatggcgg 481agaacgcggccgagagcaccgaggtgaacagccccccctcccagcccccccagcccgtcg 541tccctgctaagcccgtgcaatgcgtccatcatgtgtccactcaacccagctgcccaggac 601ggggcaagatgtccaagctgctgaacccagaggagatgacctcgagagattattacttcg 661actcctatgcccactttgggatccacgaggaaatgctgaaggatgaggtgcggactctca 721cttaccggaactccatgtaccacaacaagcacgtgttcaaggacaaagtggtactggatg 781tggggagtggtactgggatcctttccatgttcgctgccaaggcaggggccaagaaggtgt 841ttgggatcgaatgctccagtatttctgactactcagagaagatcattaaggccaaccact 901tggacaacatcatcaccatatttaagggtaaagtggaagaggtggagctgcctgtggaga 961aggtggacatcatcatcagcgagtggatgggctactgtctgttctatgagtccatgctca 1021acacggtgatctttgccagggacaagtggctgaaacctggagggcttatgtttccagacc 1081gggcagctttgtacgtggtagcgattgaagacagacagtacaaggacttcaaaatccact 1141ggtgggagaatgtctatggctttgacatgacctgcatccgggacgtggccatgaaggagc 1201ctctagtggacatcgtggatccaaagcaagtggtgaccaatgcctgtttgataaaggagg 1261tggacatttacacagtgaagacggaagagctatcgttcacatctgcattctgcctgcaga 1321tacagcgcaacgactacgtccacgccctggtcacctattttaatattgaatttaccaagt 1381gccacaagaaaatggggttttccacagcccctgatgctccctacacccactggaagcaga 1441ccgtcttctacttggaagattacctcactgtccggaggggggaggaaatctacgggacca 1501tatccatgaagccaaatgccaaaaatgtgcgagacctcgatttcacagtagacttggatt 1561ttaagggacagctgtgtgaaacatctgtatctaatgactacaaaatgcgttagcacacgt 1621gggaagctgcagagagcaacgagaaaaggaactctcacctcgatctgccgtgccgtccca 1681aagaataccgtttgcaggactacacacttgaaaaccagagttttcaactctgccttgaag 1741attggtgaactccccagggctcccgtgggctctgccactggacagaaggcctccagctcc 1801tccgctctgccctggtagcccttcacgaaggctttgtgttgccaacaaagagcgacctgg 1861cgtgctgtggctgggccccgagggtggaaacgtattcgcgtctccccgtctcctccttaa 1921ctgtgactctccgggtcttctgagttttgcatgctgcgggtgtctaggacagattgcttc 1981cactagaacctggagacatagcatctttgatagcataagccagattatctgtgtgtgcgg 2041tggtgtgcgtgtgcgtgcatgtgtgaatgtgagcagcatagttgatatttacccacaaac 2101acctgtatatgcgtgcatatacaaccaagtgggtagacctaggtgttctctcagaggggt 2161gtgtgtgtgtgtgcgtgcgcgtgtgcctagaatatatattactctcagaggagattctgt 2221tgcttttgaataggaatttgttttgtgattagttcgccccttccccaccccttaccagat 2281gttaagcagctatgaaacattctctgtactagttctggtctccttttgactggactgtgg 2341ctctgaaccttgagcatagtaccacggactccgtgggcgctcaataaacacacatgagaa 2401caaaaaaaaaaaaaaaa

[0100] An exemplary human PRMT8 amino acid sequence (PRMT8 isoform 4) is set forth below (SEQ ID NO: 22; GenBank Accession No. NP_001243465, Version NP_001243465.1 (GI:374858040), incorporated herein by reference):

TABLE-US-00006 1meslasdgfklkevssvnsppsqppqpvvpakpvqcvhhvstqpscpgrgkmskllnpee 61mtsrdyyfdsyahfgiheemlkdevrtltyrnsmyhnkhvfkdkvvldvgsgtgilsmfa 121akagakkvfgiecssisdysekiikanhldniitifkgkveevelpvekvdiiisewmgy 181clfyesmlntvifardkwlkpgglmfpdraalyvvaiedrqykdfkihwwenvygfdmtc 241irdvamkeplvdivdpkqvvtnaclikevdiytvkteelsftsafclqiqrndyvhalvt 301yfnieftkchkkmgfstapdapythwkqtvfyledyltvrrgeeiygtismkpnaknvrd 361ldftvdldfkgqlcetsvsndykmr

[0101] Although SEQ ID NO: 22 is provided as isoform 2 in the National Center for Biotechnology Information (NCBI) database, this sequence is provided as isoform 4 in FIG. 10. Isoforms 1-3 are translated from mRNA variant 1, while isoform 4 is translated from mRNA variant 2.

[0102] An exemplary human PRMT8 nucleic acid sequence (PRMT8, transcript variant 2, mRNA) is set forth below (SEQ ID NO: 23; GenBank Accession No. NM_001256536, Version NM_001256536.1 (GI:374858039), incorporated herein by reference):

TABLE-US-00007 1atttctgcaccagggaggcttgctgtttgaatgtgtgccaggttgaatggagtctctggc 61ttcagatggattcaagctgaaagaggtttcttctgtgaacagccccccctcccagccccc 121ccagcccgtcgtccctgctaagcccgtgcaatgcgtccatcatgtgtccactcaacccag 181ctgcccaggacggggcaagatgtccaagctgctgaacccagaggagatgacctcgagaga 241ttattacttcgactcctatgcccactttgggatccacgaggaaatgctgaaggatgaggt 301gcggactctcacttaccggaactccatgtaccacaacaagcacgtgttcaaggacaaagt 361ggtactggatgtggggagtggtactgggatcctttccatgttcgctgccaaggcaggggc 421caagaaggtgtttgggatcgaatgctccagtatttctgactactcagagaagatcattaa 481ggccaaccacttggacaacatcatcaccatatttaagggtaaagtggaagaggtggagct 541gcctgtggagaaggtggacatcatcatcagcgagtggatgggctactgtctgttctatga 601gtccatgctcaacacggtgatctttgccagggacaagtggctgaaacctggagggcttat 661gtttccagaccgggcagctttgtacgtggtagcgattgaagacagacagtacaaggactt 721caaaatccactggtgggagaatgtctatggctttgacatgacctgcatccgggacgtggc 781catgaaggagcctctagtggacatcgtggatccaaagcaagtggtgaccaatgcctgttt 841gataaaggaggtggacatttacacagtgaagacggaagagctatcgttcacatctgcatt 901ctgcctgcagatacagcgcaacgactacgtccacgccctggtcacctattttaatattga 961atttaccaagtgccacaagaaaatggggttttccacagcccctgatgctccctacaccca 1021ctggaagcagaccgtcttctacttggaagattacctcactgtccggaggggggaggaaat 1081ctacgggaccatatccatgaagccaaatgccaaaaatgtgcgagacctcgatttcacagt 1141agacttggattttaagggacagctgtgtgaaacatctgtatctaatgactacaaaatgcg 1201ttagcacacgtgggaagctgcagagagcaacgagaaaaggaactctcacctcgatctgcc 1261gtgccgtcccaaagaataccgtttgcaggactacacacttgaaaaccagagttttcaact 1321ctgccttgaagattggtgaactccccagggctcccgtgggctctgccactggacagaagg 1381cctccagctcctccgctctgccctggtagcccttcacgaaggctttgtgttgccaacaaa 1441gagcgacctggcgtgctgtggctgggccccgagggtggaaacgtattcgcgtctccccgt 1501ctcctccttaactgtgactctccgggtcttctgagttttgcatgctgcgggtgtctagga 1561cagattgcttccactagaacctggagacatagcatctttgatagcataagccagattatc 1621tgtgtgtgcggtggtgtgcgtgtgcgtgcatgtgtgaatgtgagcagcatagttgatatt 1681tacccacaaacacctgtatatgcgtgcatatacaaccaagtgggtagacctaggtgttct 1741ctcagaggggtgtgtgtgtgtgtgcgtgcgcgtgtgcctagaatatatattactctcaga 1801ggagattctgttgcttttgaataggaatttgttttgtgattagttcgccccttccccacc 1861ccttaccagatgttaagcagctatgaaacattctctgtactagttctggtctccttttga 1921ctggactgtggctctgaaccttgagcatagtaccacggactccgtgggcgctcaataaac 1981acacatgagaacaaa

[0103] An exemplary human PRMT8 amino acid sequence (PRMT8 isoform 2) is set forth below (SEQ ID NO: 24):

TABLE-US-00008 MKHSSRCLLLRRKMAENAAESTEVNSPPSQPPQPVVPAKPVQCVHHV STQPSCPGRGKMSKLLNPEEMTSRDYYFDSYAHFGIHEEMLKDEVRT LTYRNSMYHNKHVFKDKVVLDVGSGTGILSMFAAKAGAKKVFGIECS SISDYSEKIIKANHLDNIITIFKGKVEEVELPVEKVDIIISEWMGYC LFYESMLNTVIFARDKWLKPGGLMFPDRAALYVVAIEDRQYKDFKIH WWENVYGFDMTCIRDVAMKEPLVDIVDPKQVVTNACLIKEVDIYTVK TEELSFTSAFCLQIQRNDYVHALVTYFNIEFTKCHKKMGFSTAPDAP YTHWKQTVFYLEDYLTVRRGEEIYGTISMKPNAKNVRDLDFTVDLDF KGQLCETSVSNDYKMR

[0104] PRMT8 isoform 2 is identical to PRMT8 isoform 1; however, PRMT8 isoform 2 is truncated by 2 amino acids at the N-terminus.

[0105] An exemplary human PRMT8 amino acid sequence (PRMT8 isoform 3) is set forth below (SEQ ID NO: 25):

TABLE-US-00009 MKHSSRCLLLRRKMAENAAESTEVNSPPSQPPQPVVPAKPVQCVHHV STQPSCPGRGKMSKLLNPEEMTSRDYYFDSYAHFGIHEEMLKDEVRT LTYRNSMYHNKHVFKDKVVLDVGSGTGILSMFAAKAGAKKVFGIECS SISDYSEKIIKANHLDNIITIFKGKVEEVELPVEKVDIIISEWMGYC LFYESMLNTVIFARDKWLKPGGLMFPDRAALYVVAIEDRQYKDFKIH WWENVYGFDMTCIRDVAMKEPLVDIVDPKQVVTNACLIKEVDIYTVK TEELSFTSAFCLQIQRNDYVHALVTYFNIEFTKCHKKMGFSTAPDAP YTHWKQTVFYLEDYLTVRR

[0106] PRMT8 isoform 3 is identical to PRMT8 isoform 1; however, PRMT8 isoform 3 is truncated by 15 amino acids at the N-terminus.

PRMT8 Isoform is Essential for Cell Viability and Proliferation

[0107] Described herein is the development of a unique, reversible cell phenotype from primary human dermal fibroblasts, termed induced regeneration competent (iRC) cells. iRC cells are derived by exogenous addition of human fibroblast growth factor FGF2 and culture in reduced oxygen concentration (2%) (FIG. 6). Reduction in oxygen concentration has been shown to increase cellular lifespan and to regulate epigenetic changes (Jeltsch, A. 2013 Trends in Biochemical Sciences; Bradley et al., 1978 Journal of cellular physiology, 97: 517-522). iRC cells display increased proliferative lifespan and increased time to cellular senescence while lacking the propensity to form tumors when injected into SCID mice, a capability that is characteristic of immortalized and pluripotent cells (Page, et al., 2009 Cloning and Stem Cells, 11: 417-426; Page et al., 2011 Tissue Engineering Part A, 17: 2629-2640). This unique phenotype allows for the examination of molecular changes that lead to increased cellular lifespan without cancerous permanent self-renewal.

[0108] Small molecule inhibitors of enzymes that catalyze PTMs have been approved by the Food and Drug Administration for treatment of human cancers, and arginine methyltransferases are being hailed as the new enzymes to target for personalized cancer therapeutics (Richon, V. M., Moyer, M. P., and Copeland, R. A. (2012) Protein Methyltransferases as Targets for Personalized Cancer; Copeland et al., 2009 Nature Reviews Drug Discovery, 8: 724-732; Ott, P. A. and Adams, S. 2011 Immunotherapy, 3: 213-227; Rodrguez-Paredes, M. and Esteller, M. 2011 Nature medicine, 330-339). Prior to the invention described herein, limited evidence about PRMT regulation prevented understanding of biological consequences of corruption in their regulatory pathways. However, this family of enzymes plays a significant role in cell viability and in cancer biology (Yoshimatsu et al., 2011 International Journal of Cancer, 128: 562-573; Zakrzewicz et al., International Journal of Molecular Sciences, 13: 12383-12400; Mathioudaki et al., 2008 British Journal of Cancer, 99: 2094-2099; Goulet et al., 2007 Journal of Biological Chemistry, 282: 33009-33021; Mitchell et al., 2009 Molecular and Cellular Biology, 29: 4918-4934; Leiper, J. and Vallance, P. 1999 Cardiovascular Research, 43: 542-548; Pahlich et al., 2006 Biochimica Et Biophysica Acta-Proteins and Proteomics, 1764: 1890-1903; Wysocka et al., 2006 Frontiers in Bioscience, 11: 344-355; Pal, S. and Sif, S. 2007 Journal of Cellular Physiology, 213: 306-315; Herrmann et al., 2009 Journal of Cell Science, 122: 667-677; Di Lorenzo, A. and Bedford, M. T. 2011 Febs Letters, 585: 2024-2031; Hong et al., 2012 Biogerontology, 13: 329-336; Wang et al., 2008 Molecular and cellular biology, 28: 6262-6277; Yu et al., 2009 Molecular and Cellular Biology, 29: 2982-2996; Bedford, M. T. and Richard, S. 2005 Molecular Cell, 18: 263-272).

[0109] PRMT8 specifically has been understudied because of early reports implicating tissue specificity; however, it can no longer be ignored that PRMT8 does in fact have functional relevance outside the brain. An in vivo zebrafish study found that PRMT8 is expressed ubiquitously during early development and is critical for embryonic and neural development, as knockdown of PRMT8 resulted in early developmental defects in all three germ layers and, in many cases, death (Lin et al., 2013 PLOS ONE, 8: e55221). This was the first evidence that PRMT8 plays a critical role in development before becoming localized specifically to mature brain tissue. Described herein is PRMT8 expression in hESCs, the first evidence that PRMT8 may also function in human development. Furthermore, PRMT8 expression is demonstrated in human dermal fibroblast-derived cells, clearly indicating human PRMT8 expression outside of the CNS.

[0110] The upregulation of PRMT8 by iRC culture conditions is primarily mediated by culture in reduced oxygen, though it is potentiated by supplementation with fibroblast growth factor 2 (FGF2). Cell culture is routinely performed at atmospheric oxygen levels (between 19% to 20%) even though physiological levels tend to be much lower (ranging from 10% to 0.5%, depending on tissue type) (Dings et al., 1998 Neurosurgery, 43: 1082-1094; Harrison et al., 2002 Blood, 99: 394-394; Pasarica et al., 2009 Diabetes, 58: 718-725; Evans et al., 2006 Journal of investigative dermatology, 126: 2596-2606). As described in detail below, oxygen concentration was reduced in the model system to more closely match the physiological state. The fact that physiological oxygen levels are much lower than what is used for standard cell culture methods, and the fact that brain specifically is a hypoxic tissue (Dings et al., 1998 Neurosurgery, 43: 1082-1094), may be the cause of why PRMT8 has, until now, not been seen widely outside the CNS. It is possible that iRC culture conditions are not inducing PRMT8 expression but, rather, that standard culture conditions are repressing its expression.

[0111] The demonstration herein of increased PRMT8 protein expression with reduced oxygen is not the first indication that hypoxic conditions regulate PRMTs. In a study that analyzed PRMT 1-7 in mouse lung tissue, hypoxia was shown to be a regulator of PRMT2 (Yildirim et al., 2006 American Journal of Respiratory Cell and Molecular Biology, 35: 436-443). However, it was noted that PRMT8 was not analyzed alongside other PRMT family members in this study due to its assumed specificity to brain, highlighting the importance of recent literature that has shown PRMT8 to be ubiquitously expressed, at least during development (Lin et al., 2013 PLOS ONE, 8: e55221).

[0112] PRMT family members have variant-specific functions in various cancers, which makes them attractive targets for cancer diagnostics and/or therapeutics. For example, specific splice variants of PRMT1 demonstrate distinct activity and substrate specificity and have been correlated to tumor grade in breast cancer (Goulet et al., 2007 Journal of Biological Chemistry, 282: 33009-33021; Scott et al., 1998 Genomics, 48: 330-340; Scorilas et al., 2000 Biochemical and biophysical research communications, 278: 349-359; Mathioudaki et al., 2011 Tumor Biology, 32: 575-582). Nevertheless, the current ability to target these molecules is limited by the lack of understanding regarding expression and regulation of specific PRMT variants and the variant-specific effects they have in cancer cell lines and tumors. Prior to the invention described herein, the mechanism by which a shift from one isoform to another occurs was not known, although this shift is thought to be important for cancer development and progression. Described herein is the identification of an PRMT8 variant expressed in cells grown under iRC culture conditions, conditions that lead to increased cellular lifespan without the capacity to form tumors when injected into SCID mice (Page, et al., 2009 Cloning and Stem Cells, 11: 417-426; Page et al., 2011 Tissue Engineering Part A, 17: 2629-2640). Increased understanding about the role of PRMTs in cancer-related changes (i.e. bypassing the Hayflick limit) in a non-tumorigenic system increases understanding of PRMT regulation while offering molecular tools for development of cancer treatments and diagnostic tests.

[0113] The most interesting phenotype observed herein that PRMT8 knockdown leads to a loss of cell proliferation. As described in detail below, fibroblast transductions were performed under control conditions, with the plan to transfer to iRC conditions following selection. However, cells in knockdown treatments failed to recover following transduction, indicating that the small amount of PRMT8 present in control human dermal fibroblasts is necessary for proliferation, regardless of culture conditions. The glioblastoma line U87MG was selected for PRMT8 knockdown due to sole expression of PRMT8 variant 2. Immediate loss of proliferation in this cell type is thought to be the cause of increased sensitivity to transduction compared to primary cell types. These results encourage the continued exploration of PRMT8 as a biomarker and therapeutic target.

[0114] While other PRMTs have been robustly linked to cell cycle, this is the first evidence of PRMT8 having a functional role in cell proliferation, suggesting that PRMT8 is more similar to other PRMT family members than initially thought. In human lung fibroblasts, PRMTs 1, 4, and 6 are down-regulated as cells senesce and their expression decreases as p21 increases during senescence (Lim et al., 2008 Journal of biochemistry, 144: 523-529). In osteosarcoma, breast, bladder and lung cancer lines, PRMT1 knockdown results in G0/G1 arrest, a common hallmark of senescent cells (Yoshimatsu et al., 2011 International Journal of Cancer, 128: 562-573; Yu et al., 2009 Molecular and Cellular Biology, 29: 2982-2996; Le Romancer et al., 2008 Molecular cell, 31: 212-221). In mouse embryonic fibroblasts (MEFs), PRMT6 knockdown increases expression of both p53 and p21 (Phalke et al., 2012 Nucleic acids research, gks858; Kleinschmidt et al., 2012 PloS one 7, e41446). Because of this, it was hypothesized that the mechanism by which PRMT8 influences cell proliferation is through regulation of cell cycle. However, it remains to be determined exactly which genes and/or proteins are regulated by PRMT8.

[0115] Described herein is the upregulation of a specific gene, PRMT8, in cells which resemble pre-cancer, which may be used as a biomarker in a simple, inexpensive test as a form of preventative medicine. Also described herein is a prognostic test with pre-cancerous screening capabilities based on up-regulation of this gene.

[0116] The screen described herein takes advantage of biological samples obtained at yearly exams and physicals to screen for pre-cancerous cells, only using more invasive preventative care when necessary. A cell culture system in which cells display two-fold increase in population doublings before senescence without tumorigenesis has been described (Page et al., 2009 Cloning and Stem Cells 11, 417-426). By altering the conditions under which the cells are grown, cellular lifespan was increased more than twofold (Page et al., 2009 Cloning and Stem Cells, 11:417-426; Page et al., 2011 Tissue Engineering Part A, 17:2629-2640). Increases in cellular lifespan are relevant for the identification and characterization of biomarkers during the transformation from a healthy cell to a pre-cancer cell.

[0117] This change in phenotype has been termed extended lifespan (ELS) or induced regeneration competence (iRC), which terms are used interchangeably herein. ELS (also known as iRC) cells are used herein as a tool to characterize an early marker of increased cellular lifespan, offering potential targets for diagnostic tests. Specifically, as described in detail below, ELS cells demonstrate significant up-regulation of the arginine methyltransferase PRMT8 compared to control cells. Aberrant PRMT expression plays a role in various disease states and certain PRMT protein variants are used as prognostic markers of lung and bladder cancers (Zakrzewicz et al., 2012 International Journal of Molecular Sciences, 13:12383-12400; Yoshimatsu et al., 2011 International Journal of Cancer, 128:562-573; Mathioudaki et al., 2008 British Journal of Cancer, 99:2094-2099; and Goulet et al., 2007 Journal of Biological Chemistry, 282:33009-33021). Mutations in PRMT have been identified in skin, ovarian, and colorectal cancers (Yang Y and Bedford M T, 2013 Nature Reviews Cancer, 13:37-50).

[0118] Of relevance are deep sequencing results of cancer genomes that reveal PRMT8 to be the most mutated PRMT family member, having 15 coding region mutations out of the 106 genomes tested (Yang Y and Bedford M T, 2013 Nature Reviews Cancer, 13:37-50). In contrast, PRMT8 up-regulation in ELS cells is accompanied by increased cellular lifespan in a non-tumorigenic system. Described herein is the development of a prognostic PCR test with pre-cancerous screening capabilities based on up-regulation of PRMT8. Thus, described herein is a greater understanding of PRMT8 up-regulation within ELS cells and association with specific pre-cancer and/or cancer cell types.

[0119] Regardless of whether cancers arise as a consequence of genetic or epigenetic changes, the factors that control the balance between replicative senescence and cancerous self-renewal are of much interest as potential therapeutic targets. However, prior to the invention described herein, the molecular mechanisms that regulate this perfect balance were not well understood. As described herein, to better study this regulatory mechanism, an in vitro model system was developed which allows for increase in telomerase reverse transcriptase (TERT) levels leading to increased proliferative potential of the cells and increased time to senescence, while at the same time the cells remain non-tumorigenic when injected into severe combined immunodeficiency (SCID) mice (Page et al., 2009 Cloning and Stem Cells, 11:417-426), leading to the term extended lifespan (ELS) cells. This phenotype is also accompanied by induction of regeneration competence as demonstrated by significant reduction of collagen deposition in a mouse skeletal wound (Page et al., 2011 Tissue Engineering Part A, 17:2629-2640), leading to the term induced regeneration competent (iRC) cells to describe the cells' regenerative phenotype. As described in detail below, understanding the mechanism of cell plasticity in the context of a defined environment offers molecular tools for designing of regenerative instead of symptomatic treatment strategies.

[0120] As such, as described in detail below, it is determined whether PRMT8 is involved in increased proliferation of ELS cells by direct or indirect regulation of TERT expression, as elucidation of this pathway uncovers therapeutic targets for regenerative medicine and cancer research. As described herein, the data shows a 13.3 fold transcriptional increase in PRMT8 in ELS cells displaying nuclear localization.

[0121] Prior to the invention described herein, identification of molecular mechanisms that regulate cellular replicative lifespan was needed to better understand the transition between a normal and a neoplastic cell phenotype. As described herein, low oxygen-mediated activity of FGF2 leads to an increase in cellular lifespan and acquisition of regeneration competence in human dermal fibroblasts (iRC cells). Though cells display a more plastic developmental phenotype, they remain non-tumorigenic when injected into SCID mice (Page, et al., 2009 Cloning and Stem Cells, 11: 417-426; Page et al., 2011 Tissue Engineering Part A, 17: 2629-2640) allowing for investigation of mechanisms that regulate increased cellular lifespan in a non-tumorigenic system. As described below, analysis of chromatin modification enzymes by qRT-PCR revealed a 13.3-fold upregulation of the arginine methyltransferase PRMT8 in iRC cells. As described in detail herein, increased protein expression was confirmed in both iRC and human embryonic stem cellsthe first demonstration of endogenous human PRMT8 expression. Furthermore, as described herein, iRC cells express a PRMT8 mRNA variant. As described herein, using siRNA-mediated knockdown it was demonstrated that this variant was required for viability proliferation of human dermal fibroblasts and grade IV glioblastomas. Thus, PRMT8 upregulation in a non-tumorigenic system is a diagnostic biomarker and a therapeutic target for cells in pre-cancerous and cancerous states.

Pharmaceutical Therapeutics

[0122] For therapeutic uses, the compositions or agents described herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of a therapeutic identified herein in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the neoplasia. Generally, amounts will be in the range of those used for other agents used in the treatment of other diseases associated with neoplasia, although in certain instances lower amounts will be needed because of the increased specificity of the compound. For example, a therapeutic compound is administered at a dosage that is cytotoxic to a neoplastic cell.

Formulation of Pharmaceutical Compositions

[0123] The administration of a compound or a combination of compounds for the treatment of a neoplasia may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing a neoplasia. The compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0124] Human dosage amounts can initially be determined by extrapolating from the amount of compound used in mice, as a skilled artisan recognizes it is routine in the art to modify the dosage for humans compared to animal models. In certain embodiments it is envisioned that the dosage may vary from between about 1 g compound/Kg body weight to about 5000 mg compound/Kg body weight; or from about 5 mg/Kg body weight to about 4000 mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kg body weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg body weight; or from about 100 mg/Kg body weight to about 1000 mg/Kg body weight; or from about 150 mg/Kg body weight to about 500 mg/Kg body weight. In other cases, this dose may be about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 mg/Kg body weight. In other aspects, it is envisaged that doses may be in the range of about 5 mg compound/Kg body to about 20 mg compound/Kg body. In other embodiments, the doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight. Of course, this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient.

[0125] Pharmaceutical compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations.

Kits or Pharmaceutical Systems

[0126] The present compositions may be assembled into kits or pharmaceutical systems for use in ameliorating a neoplasia. Kits or pharmaceutical systems according to this aspect of the invention comprise a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more container means, such as vials, tubes, ampoules, or bottles. The kits or pharmaceutical systems of the invention may also comprise associated instructions for using the agents of the invention.

[0127] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, 1989); Oligonucleotide Synthesis (Gait, 1984); Animal Cell Culture (Freshney, 1987); Methods in Enzymology Handbook of Experimental Immunology (Weir, 1996); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, 1987); Current Protocols in Molecular Biology (Ausubel, 1987); PCR: The Polymerase Chain Reaction, (Mullis, 1994); Current Protocols in Immunology (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0128] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

Example 1: Regeneration Competence Accompanies Increased Expression of Arginine Methyltransferase PRMT8 in Human Adult Fibroblasts

[0129] Identification of therapeutically relevant molecules is necessary for the advancement of non-viral reprogramming of human cells for regenerative medicine. Described herein is a non-viral model system that transforms primary human dermal fibroblasts into cells with induced regeneration competence (ELS). As described in detail below, low oxygen-mediated effects of fibroblast growth factor (FGF2) lead to an increased cellular lifespan with a two fold increase in population doublings before senescence, remaining non-tumorigenic when injected into SCID mice while maintaining regeneration competence (Page et al., 2009 Cloning and Stem Cells, 11:417-426; Page et al., 2011 Tissue Engineering Part A, 17:2629-2640). This system allows for the examination of molecules that participate in increased cellular lifespan in a non-tumorigenic system. Described herein is the identification of unique molecules that contribute to the ELS phenotype with the goal to design therapeutics that target diseases associated with aging, wound healing, and tumor formation. Analysis of 84 chromatin modification enzymes by quantitative real-time polymerase chain reaction (qRT-PCR) revealed 13.3-fold upregulation of the arginine methyltransferase, PRMT8, in ELS cells. Increased protein expression was confirmed in both ELS and human embryonic stem cellsthe first demonstration of endogenous human PRMT8 expression. Also described herein is the regulation of arginine methyltransferases and the functions of endogenous PRMT8 in human cells.

[0130] Corruption of the pathway that maintains cellular senescence is associated with approximately 90% of cancers in humans (Kyo et al., 2008 Cancer Science, 99(8):1528-1538). Identification of molecules that initiate dysregulation of this pathway can be exploited for the development of cancer therapeutics. Major advancements in personalized medicine were made when terminally differentiated cells were reprogrammed into induced pluripotent stem cells (iPSCs). However, translation of this methodology for personalized medicine applications is handicapped by viral addition of reprogramming factors, low reprogramming efficiency, and tumorigenesis.

[0131] Described herein is a non-viral cell phenotype from primary human dermal fibroblasts, extended lifespan (ELS) cells. ELS cells are derived by exogenous addition of human fibroblast growth factor FGF2 and reduced oxygen concentration (2%). FGF2 is a critical component of stem cell cultures; it is a mitogen required for maintenance of pluripotency. Reduction in oxygen concentration increases cellular lifespan and regulates epigenetic changes (Jeltsch A, 2013 Trends in Biochemical Sciences, 38(4):172-176). Due to defined changes in culture conditions, ELS cells display increased population doublings, increased time to cellular senescence, and at the same time lack tumor forming capacity when injected into SCID mice (Page et al., 2009 Cloning and Stem Cells, 11:417-426). This unique phenotype allows for the examination of molecular changes that lead to increased cellular lifespan without cancerous self-renewal. At a mouse skeletal wound site, ELS cells engraft and aid in regeneration of skeletal muscle (Page et al., 2011 Tissue Engineering Part A, 17:2629-2640). Thus, culture conditions alone can induce a proregenerative, non-tumorigenic phenotype. Accordingly, a variety of biological questions regarding inhibition of senescence by environmental cues may be examined in ELS cells.

[0132] To understand molecular mechanisms that contribute to phenotypic differences between control human dermal fibroblasts and ELS cells, molecules that control epigenetic changes in adult human cells were examined. For example, arginine methyltransferases are emerging regulators of proliferation and differentiation and are established modulators of gene expression (Copeland et al., Nature Reviews Drug Discovery, 2009 8(9):724-732). Aberrant expression of protein arginine methyltransferase (PRMT) family members is associated with cardiovascular and pulmonary diseases and various types of cancers, including lung, bladder, colon, and breast cancers.

[0133] Prior to the invention described herein, little was known regarding the endogenous expression and function of PRMT8. PRMT8 has two mRNA variants transcribed from alternative 5 exons. Variant 1 has three isoforms with unique N-terminal sequences translated from differing in-frame methionines (FIG. 1). Variant 2 has only been described using genomic sequencing and is thought to have only one protein isoform. Early characterization of PRMT8 variant 1 revealed a myrostylation motif that causes sequestration to the plasma membrane. However, overexpressed PRMT8 variants 2 and 3 display nuclear localization (Kousaka et al., 2009 Neuroscience, 163(4):1146-1157). Study of PRMT8 is guided by the consensus that full-length product is endogenous and expression is restricted to brain tissue. As described herein, if endogenous PRMT8 is nuclear, it challenges the paradigm and becomes more likely that PRMT8, like other family members, has a role in critical cellular processes through chromatin modification or regulation of protein-protein interactions. Prior to the invention described herein, the expression or activity of endogenous PRMT8 in human cells was unknown.

Materials and Methods

Cell Culture

[0134] Cell culture was performed as described (Page et al., 2009 Cloning and Stem Cells, 11(3):417-426).

RT-PCR

[0135] RNA was prepared using Trizol (Invitrogen). cDNA was synthesized using qScript cDNA SuperMix (Quanta Biosciences). PCR was performed using GoTaq (Promega).

qRT-PCR Array Analysis

[0136] RNA was prepared using NucleoSpin RNA II kit (Macherey-Nagel). cDNA was synthesized using RT2 First Strand Kit (SABiosciences). Relative quantification was determined using a 7500 Real Time PCR system (Applied Biosystems) measuring SYBR green fluorescence. RT2 Profiler PCR Arrays from SABiosciences for chromatin modifying enzymes containing 84 probes were used. Fold change was calculated based on difference in Ct values.

Western Blotting

[0137] Cells were lysed by sonication. Proteins in the lysates were separated using SDS-PAGE and transferred to PVDF membranes. Antibodies used were: PRMT8 (Y. Mori; Novus NBP1-81702) and actin (Sigma A-2006). HRP-conjugated secondary antibodies were used (SantaCruz).

Immunocytochemistry

[0138] Cells were fixed 2% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Cells were blocked with 5% BSA. Alexafluor-488 labeled secondary antibody (4 m/mL, Invitrogen) was used. Nuclear counterstaining was added with 0.5 g/mL Hoechst. Antibodies used were: PRMT8 (Y. Mori; Novus NBP1-87102). Fluorescent images were acquired using confocal microscopy.

Results

[0139] To identify molecular targets that contribute to the ELS phenotype, control cells and ELS cells were harvested at day 7 to perform Human Epigenetic Chromatin Modification Enzyme Arrays (SA Biosciences). Of the 84 genes examined, the most considerable expression change was demonstrated by PRMT8, with 13.3 fold transcriptional increase in ELS cells compared to control cells (FIG. 2A).

[0140] Upregulation of PRMT8 transcript in ELS cells was detected using RT-PCR (FIG. 2B) with mouse brain cDNA as a positive control. Of note is the presence of PRMT8 transcript expression in human embryonic stem cells (hESCs).

[0141] To determine if upregulation of PRTM8 transcript correlated to upregulation of PRMT8 protein expression, Western blot analysis was performed (FIG. 2C). GST-tagged purified PRMT8 (Y. Mori) was used as a positive control. The 26 kD GST tag is responsible for the shift of PRMT8 from 45 kD to 71 kD. These results also demonstrate endogenous PRMT8 protein expression in hESCs for the first time.

[0142] To explore the subcellular localization of endogenous PRMT8 in human cells, immunocytochemistry (ICC) was employed (FIG. 3). PRMT8 localization was restricted almost exclusively to nuclei as PRMT8 expression colocalized with Hoechst nuclear stain.

[0143] These data suggest endogenous PRMT8 expressed in ELS cells is likely not the myristoylated full-length isoform, providing similarities between human PRMT8 and reports of endogenous mouse PRMT8 (Kousaka et al., 2009 Neuroscience, 163(4):1146-1157). This work also supports evidence for PRMT8 function outside of the nervous system with a potential role in development. Described in detail below is the functional role of PRMT8 in relation to increased lifespan of ELS cells using its overexpression and knockdown.

Example 2: Identification of the PRMT8 Variant Up-Regulated in ELS Cells

[0144] As described above, PRMT8 is up-regulated in ELS cells compared to control cells at both the transcript and protein level (FIG. 2B and FIG. 2C). Variant specific expression of PRMT1, the family member most similar to PRMT8, increases during progression of tumor formation in colon cancer (Mathioudaki et al., 2008 British Journal of Cancer, 99: 2094-2099). Because of the importance of PRMT variant expression in disease states, the variant identity of ELS-PRMT8 is determined as described herein. Two mRNA variants of PRMT8 have been described with a third mRNA sequence predicted. Human embryonic stem cells (hESCs) and ELS cells were tested by PCR for mRNA variants by variant specific PCR. After analysis, neither variant 2 is present in ELS cells (FIG. 4). The importance of ELS-PRMT8 variant identification for use as a biomarker is underscored by the prevalence of PRMT8 mutations in skin, ovarian, and colorectal cancers (Yang Y. and Bedford M. T., 2013 Nature Reviews Cancer, 13:37-50).

[0145] PRMT8 variant identification is carried out in two ways: 1) 5 Rapid Amplification of cDNA Ends (RACE) to identify the mRNA variant present in ELS cells, and 2) LC/MS to sequence the protein isoform present in ELS cells. A 5 RACE System (Life Technologies) would enable the identification of the mRNA variant of PRMT8 present in ELS cells. PRMT8 specific primers are purchased from Integrated DNA Technologies (IDT). PRMT8 antibody is purchased from Novus Biologicals. Finally, precast polyacrylamide gels are purchased from BioRad.

Example 3: The Role of PRMT8 in ELS Cells on the ELS Phenotype

[0146] ELS cells demonstrate significant increase in cellular lifespan: while control fibroblasts undergo 33 population doublings over 59 days, ELS cells undergo 68 population doublings over 76 days (Page et al., 2009 Cloning and Stem Cells, 11:417-426). Cellular senescence is critical for maintaining genomic integrity; corruption of the pathway that maintains cellular senescence is associated with approximately 90% of cancers in humans (Kyo et al., 2008 Cancer Science, 99: 1528-1538). Prior to the invention described herein, there have been no reports addressing the effect of PRMT8 expression on cellular senescence. However, an increasing number of publications are identifying roles for other PRMT family members in senescence regulation. In human fibroblasts, PRMT1 protein levels decrease significantly as cells reach replicative senescence (Lim et al., 2008 Journal of biochemistry, 144:523-529). Factors that regulate loss of PRMT1 over the course of cellular lifespan appear to be critical for cellular senescence. PRMT1 is up-regulated in lung and bladder cancer, where abrogation of PRMT1 suppresses cancer cell growth (Yoshimatsu et al., 2011 International Journal of Cancer, 128:562-573). This suggests corruption of the pathway that maintains cellular senescence is accompanied by increased PRMT1 expression.

[0147] Prior to the invention described herein, a role for PRMT8 in cellular senescence had not been identified. Described herein is the characterization of the role of PRMT8 on increased lifespan in a non-tumorigenic system.

[0148] A protocol is developed that measures differences in telomere length between ELS and control cells, a potential indicator for changes in cellular senescence. This optimized method is utilized to determine if PRMT8 overexpression or knockdown affects telomere length and/or telomerase activity. The role of PRMT8 on the ELS phenotype is assessed with both loss-of-function and gain-of-function experiments.

[0149] For loss-of-function, lentiviral particles against PRMT8 were purchased from GenTarget Inc. Current experiments are being done to optimize PRMT8 knockdown (FIG. 5A-FIG. 5C). After transduction, primary cells are moved to ELS conditions until senescence. Lentiviral overexpression particles are purchased from transOMIC.

[0150] For gain-of-function, the PRMT8 protein sequence from mRNA variant 2 is utilized. Lentiviral particles overexpressing ELS-PRMT8 with a C-terminal GFP-tag and Puromycin resistance are developed (transOMIC). PRMT8 is overexpressed in primary human dermal fibroblasts and overexpression is confirmed with Western blotting. To maintain stable overexpression PRMT8 cell lines, Puromycin selection is used to select for PRMT8 integration. After transfection, primary cells are kept in control conditions until senescence.

[0151] As a readout for the ELS phenotype, population doublings and time to cellular senescence is measured in overexpression and knockdown PRMT8 cells. Cells are seeded at a density of 16,000 cells per well of a 24 well plate at each passage. Cultures are maintained in appropriate conditions (either control or ELS) until cells senesce. Senescence is determined as the first calculation of negative population doublings and is confirmed with flow cytometry analysis of senescence associated -galactosidase, the most widely used biomarker for senescent cells. Population doublings are calculated as log 2 (final cell count/initial cell count).

Example 4: PRMT8 Expression Panel of Cancer Cell Lines

[0152] A prognostic test will require correlation of ELS-PRMT8 up-regulation with specific types of pre-cancer and cancer cell types. First, literature is reviewed for up-regulation of PRMT8 in various pre-cancer and cancer type(s). Second, PRMT8 up-regulation is examined in cell lines associated with identified pre-cancer and cancer types(s) by RT-PCR. Finally, various primary tissue types from identified pre-cancers and cancers are examined for up-regulation of ELS-PRMT8 by RT-PCR.

[0153] To obtain preliminary data regarding the up-regulation of PRMT8 transcript and its potential association with specific types of pre-cancers and cancers, NCBI and COSMIC (Catalog of Somatic Mutations in Cancer) databases, which curate published gene expression profiles, are reviewed. This provides an inexpensive way to rule out a variety of different cell types from the analysis based on previous experimentation.

[0154] When at least one viable cell type is targeted based on previously published data, cell lines corresponding to that specific pre-cancer/cancer type are obtained and tested for ELS-PRMT8 up-regulation with RT-PCR. Focus is placed on cell types that can be obtained using non-invasive methods typically performed during routine physicals, such as blood, stool, or urine collection.

[0155] For cell types that demonstrate up-regulation of ELS-PRMT8, a larger sample pool is obtained to determine if the ELS-PRMT8 up-regulation is a common molecular mark of that pre-cancer/cancer type. Samples are obtained from BioServe, a tissue repository of more than 600,000 primary samples from more than 120,000 patients. PRMT8 is considered a biomarker for any per-cancer or cancer type that demonstrates increased PRMT8 expression for a significant number of samples tested compared to patient matched control tissue.

Example 5: Arginine Methyltransferase 8 Isoform is Essential for Cell Viability Proliferation

[0156] As described above, aberrant arginine methyltransferase expression is correlated to various cancers. As described in detail below, culture conditions that increase lifespan without tumorigenesis induce expression of a variant of arginine methyltransferase, PRMT8. As described below, this PRMT8 variant is required for cell proliferation. Indeed, molecules that regulate the balance between senescence and unregulated proliferation (e.g., PRMT8) may be indicative of early pre-cancer cells.

Materials and Methods

[0157] The following materials and methods were utilized in this example.

Cell Culture

[0158] The adult human fibroblast line CRL-2352 was obtained from American Tissue Culture Collection (ATCC; Manassas, Va.) at passage 2. The foreskin fibroblast line CRL-2097 was obtained from ATCC. The adult human fibroblast line CT-1005 was obtained from a panniculectomy at UMass Medical (Worcester, Mass.) through their tissue distribution program. Cells were cultured in medium consisting of DMEM: Ham's F12 (50:50; MediaTech) with 10% Fetal Clone III (HyClone). The DMEM (without L-Gln or phenol red) was supplemented with 4 mM fresh L-Gln (MediaTech, Manassas, Va.) prior to use. Cultures were carried out in a 37 C. incubator in a humidified environment of 5% CO.sub.2 and either 19% or 2% 02 depending on experimental requirement. All cultures were processed for analyses on day 7. When used, media was supplemented with human recombinant FGF2 (PeproTech) at 4 ng/mL. Human embryonic stem cellshESCs (W09; WiCell, Madison, Wis.) were cultured on mytomycin C-treated mouse embryonic fibroblasts seeded onto 0.1% gelatin coated six-well plates using 80% Knockout DMEM (Invitrogen), 20% Knockout serum replacement supplemented with 2.0 mM L-Gln, 0.055 mM 2-mercaptoethanol, and 4.0 ng/mL FGF2, as recommended by the supplier. Glioblastomas (U87MG; ATCC) were cultured in medium consisting of DMEM: Ham's F12 (50:50; MediaTech) with 10% Fetal Clone III (HyClone).

RT-PCR

[0159] RNA was prepared by Trizol (Invitrogen, Inc.) according to the manufacturer's instructions and quantified by spectrophotometry (NanoDrop 2000). One microgram of total RNA was used to perform first strand cDNA synthesis using qScript cDNA SuperMix (Quanta Biosciences). Mouse brain RNA was a generous gift from RXi Pharmaceuticals. For RT-PCR, 50 ng first-strand cDNA was used as a template for each reaction. PCR was performed using 12.5 L GoTaq (Promega) and 0.2 mM each of forward and reverse primers for PRMT1, PRMT8, PRMT8 variant 1, PRMT8 variant 2, GFP, and actin (Table 2). PCR products from the primary round of amplification were diluted 1:100 with Tris EDTA and the diluted primary PCR product was used as product for the second round of amplification of PRMT8 variant 2 by nested PCR. Amplification products were resolved on 2% agarose gels containing 0.5 g/mL ethidium bromide in 1TAE buffer and photographed using a BioRad Gel Doc XR System.

qRT-PCR Array Analysis

[0160] RNA was prepared using NucleoSpin RNA II kit (Macherey-Nagel) according to the manufacturer's instructions and quantified by spectrophotometry (NanoDrop 2000). Two micrograms of total RNA was used to perform first strand cDNA synthesis using RT2 First Strand Kit (SABiosciences) as recommended by the supplier. Relative quantification was determined using a 7500 Real Time PCR system (Applied Biosystems, Bedford, Mass.) measuring SYBR green fluorescence (RT2 SYBR Green/ROX qPCR Master Mix, SABiosciences). RT2 Profiler PCR Arrays from SABiosciences for chromatin modifying enzymes containing 84 probes were used to identify genes with altered expression in the presence of FGF2 and when oxygen levels were reduced. Analysis was performed by SABiosciences RT2 Profiler PCR Array Data Analysis Template v3.3. Fold change was calculated based on difference in Ct values.

Cloning

[0161] PRMT8 was amplified using RT-PCR described above. The PCR product was resolved on a 2% agarose gel and the 205 bp band was excised and cleaned using a NucleoSpin Gel and PCR Clean-up column (Macherey Nagel) according to the manufacturer's instructions. A Klenow (New England Biolabs) reaction was performed using the entire PCR product. The reaction was incubated at room temperature for 15 minutes then stopped with the addition of 10 M EDTA, followed by a column clean up (NucleoSpin, Macherey Nagel). 70 ng from the Klenow reaction were treated with T4 kinase (New England Biolabs). The kinase reaction was incubated at 37 C. before cleaning over a column (NucleoSpin, Macherey Nagel). A T4 ligation was performed with 20 ng pLVX-puromycin (Clontech Laboratories, Inc.) and PCR product in a 1:1 ratio overnight at 4 C. 10 L of ligated pLVX was then transformed into chemically competent E. coli cells. Transformants were incubated on ice for 30 minutes and heat shocked at 42 C. for 45 seconds before 250 L S.O.C. media was added. Transformants were incubated at 37 C. for 1 hour with agitation prior to overnight incubation on puromycin-containing agar plates at 37 C. Colonies were picked and plasmids were cultured in 3 mL LB broth containing ampicillin overnight with agitation at 37 C. Minipreps were performed on plasmid cultures using a NucleoSpin Plasmid Kit (Macherey Nagel) according to the manufacturer's instructions. Insertion of the PCR product was confirmed with a double restriction digest using 500 ng DNA, 5 units ClaI (New England Biolabs), and 5 units BamHI (New England Biolabs) prior to sequencing (GeneWiz, Cambridge, Mass.).

5 Rapid Amplification of cDNA Ends

[0162] 5 sequences were determined using a 5 RACE System for Rapid Amplification of cDNA Ends kit (Invitrogen) according to the manufacturer's instructions. Briefly, cDNA was synthesized using a primer specific to PRMT8 (5-CGAGACCTCGATTTCACAG (SEQ ID NO: 9)), the sample was purified over a column, and the enzyme terminal deoxynucleotidyl transferase (TdT) was used to add a series of cytosine residues to the 3 end of the product. Nested PCR was then performed, the products were run on a 1.5% agarose gel, and bands were excised, purified (Macherey Nagel; Nucleospin Extract II), and sequenced (GeneWiz, Cambridge, Mass.). Primer sequences for nested amplification are as follows: primary PCR-forward primer provided by Invitrogen (abridged anchor primer); reverse primer 5-CTTGGCAGCGAACATGGAAA (SEQ ID NO: 10) (hES), 5-CACCAGTGGATTTTGAAGTCCTTG (SEQ ID NO: 11) (iRC); nested PCR-forward primer provided by Invitrogen (abridged universal amplification primer); reverse primer 5-CATCCAGTACCACTTTGTCCT (SEQ ID NO: 12)(hES), 5-CTGGAAACATAAGCCCTCCAGG (SEQ ID NO: 13) (iRC).

Transduction

[0163] Custom lentiviral particles were designed and produced by GenTarget Inc. (San Diego, Calif.) to target PRMT8 for knockdown using shRNA. Particles contained shRNA constructs driven by an H1 promoter with a GFPpuromycin reporter tag driven by an RSV promoter. Human dermal fibroblasts were seeded at 1.610.sup.4 cells per well of a 12 well plate and incubated at 37 C. overnight. Media was removed and 0.4 mL serum-free media was added to each treatment well, followed by lentiviral particles to a multiplicity of infection of 50. Cells were incubated at 37 C. for 6 hours. Six hours post-transduction, 1 mL complete media was added to each well. Cells were imaged every 24 hours for GFP expression and cumulative population doublings were determined via cell counts. Glioblastomas were seeded at 4.010.sup.4 cells per well of a 6 well plate and transduced with lentiviral particles to a multiplicity of infection of 50. Transfection efficiency was monitored by expression of GFP on a Zeiss inverted epifluorescence microscope (Axiovert 200M) using AxioVision software (AxioVs40 V 4.8.2.0, service pack 4.8.2 SP1). All images were obtained with an AxioCam MRm camera using a 20LD Plan-Neofluar objective (20/0.4 Ph2 Korr) using identical settings.

Protein Isolation and Western Blotting

[0164] Total protein was isolated from subconfluent cells with cell lysis buffer (200 mM Tris-HCl; pH 7.5, 750 mM NaCl, 40% glycerol, 0.0626% Trition-X 100, 0.025% Tween-20, 0.1% NP-40), supplemented with compete protease inhibitor cocktail (PIC, Santa Cruz Biotechnology). Lysis was performed using sonication (Misonix XL2000) on power 3 with 5 pulses performed 3 times. Protein concentration was determined using Coomassie (Bradford) Protein Assay Kit (Thermo Scientific). Protein supernatant and 5 loading dye (10% SDS, 40% glycerol, 1% Bromophenol Blue, 31.3% 1M Tris-HCl; pH 6.8, 5% 2-13mercaptoethanol) were mixed in a 5:1 ratio and boiled for 5 minutes. Proteins were separated using 12% SDS-PAGE at indicated concentrations of total protein in the lysate and transferred to PVDF membranes (BioRad Laboratories) using Towbin transfer buffer (25 mM Tris Base, 192 mM glycine, 20% methanol, 0.037% SDS). The membranes were blocked with Tween-Tris-buffered saline (TBST: 25 mM Tris Base, 137 mM NaCl, 2.7 mM KCl, 0.2% Tween-20) and 5% dry milk while shaking at room temperature for 60 minutes. Primary antibodies were incubated with the membrane in TBST and 1% dry milk rotating overnight at 4 C.: antiPRMT8 (Novus NBP1-81702; 1:200) and anti-actin (Sigma A-2066; 1:5000). HRPconjugated secondary antibodies (SantaCruz Biotechnologies) were incubated with the membrane in TBST and 1% dry milk rotating at room temperature for 2 hours. Between and after antibody incubations, membranes were washed 4 times for 10 minutes each with TBST. Chemilluminescence signal was developed by luminol (SantaCruz Biotechnolgies) and luminescence detected using a BioRad Gel Doc XR System. Densitometry was used for quantitation of the signal. Obtained values (N=3) were normalized to actin and means compared using one-tailed T-test. Difference between the means was judged at p<0.05.

Results

PRMT8 is Expressed in Human Dermal Fibroblasts

[0165] In an effort to understand molecular mechanisms associated with increased lifespan in iRC cells, known epigenetic modulators were examined as potential candidates. To identify possible target genes, expression of 84 chromatin modification enzymes were analyzed by an qRT-PCR array in fibroblasts grown under control and iRC culture conditions (n=1). Expression was normalized to the housekeeping gene that showed the least divergent expression between experimental groups, the ribosomal protein RPL13. Fold change was calculated by comparing Ct values between treatment groups (Ct). FIG. 7A shows the top 5 most up- and down-regulated genes, per the array, in iRC cells compared to cells grown in the absence of exogenous FGF2 and at ambient oxygen. Of the 84 genes examined, the most considerable expression change was observed in protein arginine methyltransferase 8 (PRMT8), represented by a 13.3-fold transcriptional increase in iRC cells compared to control cells. Expression levels of all other arginine methyltransferases remained relatively unchanged between culture conditions (FIG. 7B). Expression of the most recently-described PRMT family member, PRMT9, was not assessed.

[0166] As the array was merely used to identify potential targets for study, PRMT8 was examined as a gene of interest using RT-PCR and Western blotting. Primers were designed to recognize the region of PRMT8 analyzed in the chromatin modification enzyme array. Careful consideration was given during primer design due to high homology between PRMT8 and others within the PRMT family, especially PRMT1 (Lee et al., 2005 Journal of Biological Chemistry, 280: 32890-32896; Hung, C. M. and Li, C. 2004 Gene, 340: 179-187; Lin et al., 2013 PLOS ONE, 8: e55221; Sayegh et al., 2007 Journal of Biological Chemistry, 282: 36444-36453 Kousaka et al., 2009 Neuroscience, 163: 1146-1157). Upregulation of PRMT8 transcript in iRC cells was validated by RT-PCR (FIG. 8A) using mouse brain cDNA as a positive control. Curiously, PRMT8 was also expressed in human embryonic stem cells (hESCs). To determine if upregulation of PRMT8 transcript is accompanied by upregulation of PRMT8 protein expression, Western blot analysis was performed (FIG. 8B). The immunogenic protein was detected at the expected 45 kDa in both iRC and hESC cells. The control PRMT8 protein migrated at 7 lkDa, due to its 26 kDa GST tag. FIG. 8B is a representative blot; densitometry for 3 replicates can be seen in FIG. 8C. All samples were normalized to actin and analyzed using a onetailed T-test.

[0167] As iRC cells are derived from primary human dermal fibroblasts, any sample is subject to individual idiosyncrasies in gene expression. Because of this, PRMT8 expression was analyzed in other primary human dermal fibroblast lines to ensure PRMT8 upregulation is a result of iRC culture conditions and not an artifact of the individual from whom the fibroblasts were derived. Though previous and subsequent work was carried out using CRL-2352s (human adult dermal fibroblasts), other cells, specifically CRL2097s (human foreskin fibroblasts) and CT-1005s (adult female panniculectomy fibroblasts), also demonstrated upregulation of PRMT8 by RT-PCR when grown under iRC culture conditions (FIG. 8D). The significance of low expression in young human tissue (CRL-2097s) compared to high expression in mature human tissue (CRL2352s and CRL-1005s) is not clear. However, even in young tissue expression appears to be inducible by iRC conditions, supporting the idea that regulation of PRMT8 is not dependent on organismal age.

[0168] Since consensus opinion relegates expression of PRMT8 strictly to brain tissue (Lee et al., 2005 Endocrine reviews, 26: 147-170; Sayegh et al., 2007 Journal of Biological Chemistry, 282: 36444-36453; Taneda et al., 2007 Brain Research, 1155: 1-9), the transcript detected in iRC cells was sequenced in order to verify the identity of the transcript as PRMT8. PRMT8 was amplified from iRC cells using RT-PCR, the band was excised from the gel, and the fragment was cloned into pLVX using T4 ligase. Positive transformants were sent for sequencing, and the identity of the transcript was verified as that of PRMT8 (FIG. 9).

Human Dermal Fibroblasts Express a PRMT8 Variant

[0169] Aberrant PRMT expression plays a role in various disease states, and certain PRMT protein variants are used as prognostic markers for lung and bladder cancers (Yoshimatsu et al., 2011 International Journal of Cancer, 128: 562-573; Zakrzewicz et al., International Journal of Molecular Sciences, 13: 12383-12400; Mathioudaki et al., 2008 British Journal of Cancer, 99: 2094-2099; Goulet et al., 2007 Journal of Biological Chemistry, 282: 33009-33021). As such, it is critical to understand variant and isoform expression of this family of enzymes for development and improvement of diagnostic and therapeutic tools. Through genomic sequencing, a second mRNA variant for PRMT8 was identified (NM_001256536)one transcribed from an alternate 5 exon (FIG. 10A). Between mRNA variants 1 and 2, only exon 1 differs, while exons 2 through 10 are identical. FIG. 10B illustrates primer locations for both mRNA variants used for sequence determination by 5 Rapid Amplification of cDNA Ends (RACE) and RT-PCR. Also shown are the 4 PRMT8 protein isoforms along with their experimentally determined subcellular localizations. Prior to the invention described herein, only mRNA variant 1 (NM_019854) (incorporated herein by reference) has been studied, along with the 3 protein isoforms translated from that variant (Lee et al., 2005 Journal of Biological Chemistry, 280: 32890-32896; Sayegh et al., 2007 Journal of Biological Chemistry, 282: 36444-36453; Kousaka et al., 2009 Neuroscience, 163: 1146-1157). Isoform 1 harbors a myristoylated residue, conferring plasma membrane localization, while isoforms 2 and 3 are truncated at the N-terminus and lack the myristoylation motif, resulting in nuclear localization (Kousaka et al., 2009 Neuroscience, 163: 1146-1157). 5 RACE was used to reveal which PRMT8 mRNA variants are expressed in both hESCs and iRC cells (FIG. 11A-FIG. 11B). 5 RACE with hESC cDNA produced a band that, when sequenced, was identical to exon 1 of PRMT8 mRNA variant 1, beginning with the 172nd nucleotide. 5 RACE with iRC cell cDNA produced a band that, when sequenced, was identical to exon 1 of PRMT8 mRNA variant 2, beginning with the 1st nucleotide (FIG. 11C). In the National Center for Biotechnology Information (NCBI) database, the sequence for PRMT8 variant 2 was predicted by a combination of genomic DNA and transcript sequences. The cDNA sequence for PRMT8 variant 2 has been curated by NCBI and can be found via the accession number KR014345, incorporated herein by reference.

[0170] Forward primers were designed to amplify a region on exon 1 of either variant 1 or variant 2 (FIG. 10A-FIG. 10B) to develop variant-specific PCR so that RT-PCR could be used to test PRMT8 variant expression in other cell types (FIG. 11D). Variant 2 amplification required semi-nested PCR. Sequences for variant-specific primers are presented in Table 2. RT-PCR demonstrated that both control fibroblasts and iRC cells express PRMT8 variant 2 transcript, with control cells expressing low levels, while hESCs express PRMT8 variant 1 transcript, validating the 5 RACE data.

TABLE-US-00010 TABLE2 DNAprimersequenesforRT-PCR Fwdprimer Revprimer Amplicon Primer (5 to3) (5 to3) (bp) PRMT1 CTCTGGTATA GCTCATCCCAT 149 AGGCGGTCCC TAGCCAAGGT (SEQIDNO:14) (SEQIDNO:19) PRMT8 GACTACGTCCAC GGTCTCGCACAT 205 GCCCTGGTCACC TTTTGGCATTTG TATTTTATT GCTTCATGG (SEQIDNO:1) (SEQIDNO:5) PRMT8 AAGGAATCCGGA GGCATAGGAGTCGA 458 v1 GCAGATGAGAAG AGTAATAATCTCTC (SEQIDNO:2) (SEQIDNO:6) PRMT8 CTGTTTGAATGT GGCATAGGAGTCGA 240 v2 GTGCCAGGTTG AGTAATAATCTCTC (SEQIDNO:3) (SEQIDNO:6) PRMT8 TGAATGTGTGCCA GGCATAGGAGTCGA 235 v2 GGTTGAATGGAG AGTAATAATCTCTC nested (SEQIDNO:4) (SEQIDNO:6) GFP AGCTGACCCTG CTGCTTGTCGGC 350 AAGTTCATCTG CATGATATAGAC (SEQIDNO:15) (SEQIDNO:18) Actin TCTGGCACCAC CTTCTCCTTAA 392 ACCTTCTACAA TGTCACGCACG (SEQIDNO:16) (SEQIDNO:19)

PRMT8 Variant 2 is Critical for Proliferation of Human Dermal Fibroblasts

[0171] The iRC phenotype is, in part, characterized by increased cellular lifespan. Because of this, it was next examined whether there is a causal link between increased lifespan and upregulation of PRMT8. PRMT8 was knocked down using custom lentiviral particles containing shRNA constructs designed to target both known mRNA variants of PRMT8-shRNA vector #1 targets PRMT8 within exon 4, shRNA #2 within exon 6, and shRNA #3 within exon 9.

[0172] To demonstrate knockdown success and specificity, the glioblastoma line U87MG was transduced with each shRNA construct separately, including the scramble control, and cells were imaged and then harvested 2 days post-transduction for analysis by RT-PCR. Microscopy demonstrated all treatment groups transduced with the scramble control construct, shRNA #1 (ACCACTTGGACAACATCATCAcgagTGATGATGTTGTCCAAGTGGT (SEQ ID NO: 26), shRNA #2 (AGCTTTGTACGTGGTAGCGATcgagATCGCTACCACGTACAAAGCT (SEQ ID NO: 27), and shRNA #3 (GGAAGCAGACCGTCTTCTACTcgagAGTAGAAGACGGTCTGCTTCC (SEQ ID NO: 28), express the GFP reporter, indicating successful transduction in all treatments (FIG. 12A) RT-PCR demonstrated successful PRMT8 knockdown, as control and scramble control treatments express PRMT8 variant 2 transcript, while knockdown treatments do not at detectable levels (FIG. 12B). In each of SEQ ID NOs: 26-28, capital letters to the left comprise the sense strand; lower case letters in the middle comprise the loop; and capital letters to the right comprise the anti-sense strand. Based on this data, shRNA construct #2 was selected for use in experiments to determine the effect of PRMT8 on cellular lifespan. PRMT1 was used to determine knockdown specificity since PRMT8 and PRMT1 are the most homologous members of the PRMT family, sharing over 80% sequence identity (Lee et al., 2005 Journal of Biological Chemistry, 280: 32890-32896; Sayegh et al., 2007 Journal of Biological Chemistry, 282: 36444-36453; Kousaka et al., 2009 Neuroscience, 163: 1146-1157).

[0173] Fibroblasts were thawed at passage 7 and transduced at day 0 (FIG. 13A). Puromycin selection pressure was applied for 7 days to all treatment groups except control cells, beginning 3 days after transduction. Control cells reached an average of 13.8 PDs on day 42 post-transduction and control cells receiving scrambled shRNA reached an average of 9.79 cumulative PDs after selection recovery on day 42 post-transduction. Reduced viability of cells transduced with scrambled shRNA is likely due to cell type specific effects of transduction on primary cells. It has long been known that primary cells are notoriously difficult to transduce due to low efficiency and excessive cell deatha consequence not observed with immortalized cells (Halbert et al., 1995 Journal of virology, 69: 1473-1479). Cells transduced with PRMT8 shRNA reached an average of 1.26 PDs on day 42 posttransduction with a peak of 0.63 PDs on day 6 post-transduction. Transduction efficiency was monitored by expression of a GFP reporter and GFP fluorescence was imaged weekly throughout the study. FIG. 13B shows representative images from each treatment on day 6 posttransduction and day 14 post-transduction.

PRMT8 is Critical for Proliferation of Grade IV Glioblastomas

[0174] The validity of PRMT8 as a pre-cancer biomarker requires demonstration of the necessity of this gene for proliferation of preneoplastic as well as tumorigenic cells. Accordingly, PRMT8 was next knocked down in glioblastomas to determine whether PRMT8 expression is required for proliferation of this highly aggressive cancer. The glioblastoma line U87MG was transduced at day 0 and puromycin selection pressure was applied for 3 days to all treatment groups except control cells beginning 3 days after transduction (FIG. 14A). Again, three replicates were performed and cumulative PDs were averaged. Control cells reached an average of 4.89 cumulative PDs on day 16 post-transduction. Cells transduced with scramble control shRNA reached an average of 2.83 cumulative PDs on day 16 post-transduction. Cells transduced with PRMT8 shRNA reached an average of 3.73 cumulative PDs on day 6 posttransduction. No data was recorded for PRMT8 shRNA treated cells on day 15 as all cells within the treatment were dead. The experiment was terminated after day 16 due to complete death in the PRMT8 shRNA treatment group. Transduction efficiency was monitored with a GFP reporter and GFP expression in all treatments from day 1 and day 6 can be seen in FIG. 14B.

Other Embodiments

[0175] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0176] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference.

[0177] Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[0178] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.