ENERGETIC CANCER STEM CELLS (E-CSCS): A NEW HYPER-METABOLIC AND PROLIFERATIVE TUMOR CELL PHENOTYPE, DRIVEN BY MITOCHONDRIAL ENERGY
20230040965 · 2023-02-09
Inventors
- Michael P. LISANTI (Didsbury Village Greater Manchester, GB)
- Federica SOTGIA (Didsbury Village Greater Manchester, GB)
- Marco FIORILLO (Manchester, GB)
Cpc classification
A61K31/519
HUMAN NECESSITIES
C12Q2600/106
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
G01N2800/52
PHYSICS
C12Q2600/112
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K31/519
HUMAN NECESSITIES
Abstract
This disclosure describes the characteristics of the “energetic” cancer stem cell (e-CSC) phenotype. This distinct sub-population of cancer stem cells (CSCs) has a unique energetic profile compared to bulk CSCs, being more glycolitic, having higher mitochondrial mass and elevated oxidative metabolism. e-CSCs also show an increased capacity to undergo cell cycle progression, enhanced anchorage-independent growth, and ALDH-positivity. The e-CSC phenotype presents new targets for cancer therapeutics, and in particular the anti-oxidant response, mitochondrial energy production, and mitochondrial biogenesis of e-CSCs makes them highly susceptible to mitochondrial inhibitors that target e-CSC anti-oxidant response, mitochondrial energy production, and mitochondrial biogenesis. Gene products for e-CSCs are disclosed, as well as classes of mitochondrial inhibiting therapeutic agents. Also disclosed are methods for identifying and separating e-CSCs front bulk cell populations.
Claims
1. A method for predicting and treating tamoxifen resistance in a breast cancer, the method comprising: obtaining a biological sample of the cancer; determining, or having determined, a level of expression of each member of an e-CSC gene signature comprising aldehyde dehydrogenase 5 family member A1 (ALDH5A1), thioredoxin reductase 1 (TXNR), and ribonucleotide-diphosphate reductase subunit M2 (RRM2); comparing the determined level to a threshold level from a healthy cell line for each member of the e-CSC gene signature; administering a pharmaceutically effective amount of at least one of an oxidative phosphorylation (OXPHOS) inhibitor and a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor if the determined level exceeds the threshold level.
2. The method of claim 1, wherein the gene signature further comprises NAD(P)H quinone dehydrogenase 1 (NQO1).
3. The method of claim 1, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor comprises diphenyleneiodonium chloride (DPI).
4. The method of claim 1, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor comprises Ribociclib.
5. The method of claim 1, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor comprises at least one of DPI, atovaquone, irinotecan, sorafenib, niclosamide, berberine chloride, Ribociclib, Abemaciclib, and Palbociclib.
6. The method of claim 1, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor comprises the combination of at least one OXPHOS inhibitor and at least one CDK4/6 inhibitor.
7. The method of claim 1, wherein the pharmaceutically effective amount of at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor is administered if the quotient of the determined level divided by the threshold level is greater than 1.2.
8. A method for predicting and treating tamoxifen resistance in a breast cancer, the method comprising: obtaining a biological sample of the cancer; determining, or having determined, a level of expression of each member of an e-CSC gene signature comprising NAD(P)H quinone dehydrogenase 1 (NQO1), aldehyde dehydrogenase 5 family member A1 (ALDH5A1), thioredoxin reductase 1 (TXNR), and ribonucleotide-diphosphate reductase subunit M2 (RRM2); comparing the determined level to a threshold level from a healthy cell line for each member of the e-CSC gene signature; administering a pharmaceutically effective amount of at least one of an oxidative phosphorylation (OXPHOS) inhibitor and a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor if the determined level exceeds the threshold level.
Description
DESCRIPTION OF THE DRAWINGS
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DESCRIPTION
[0035] The following description includes the currently contemplated modes of currying out exemplary embodiments of the present approach. The following description is not to be taken in a limiting sense, and is made merely for the purpose of illustrating the general principles of the invention.
[0036] As described herein, the present approach relates to identifying, purifying, and collecting a hyper-proliferative cell sub-population of breast CSCs, by using an endogenous marker of energy-metabolism, namely, flavin-derived auto-fluorescence. The present approach may take various forms, depending on the embodiment. For example, under the present approach, a gene signature is provided for detecting the presence of e-CSCs, predicting tumor recurrence, and/or predicting metastasis. The present approach also allows for purifying and collecting e-CSCs from a sample. In some embodiments, the present approach allows for treating cancer through targeting and/or eradicating e-CSCs in a mass.
[0037] In addition to having a hyper-proliferative phenotype, e-CSCs showed progressive increases in stemness markers (e.g., ALDH activity and mammosphere-forming activity), a highly elevated mitochondrial mass, as well as increased glycolytic and mitochondrial activity. Moreover, the 3D sub-type of e-CSCs is strictly dependent on mitochondria, for cell propagation. Thus, under the present approach, the anchorage-independent propagation e-CSCs, derived from 3D-spheroids, may be specifically targeted with an OXPHOS inhibitor (such as, for example, DPI) to inhibit mitochondrial biogenesis, and/or a CDK4/6 inhibitor (such as, for example, Ribociclib) to inhibit cell proliferation.
[0038] Mechanistically, there are at least 2 different classes of e-CSCs that are metabolically distinct. As used herein, M-H refers to “monolayer-high” cells, and S-H refers to “spheroid-high” cells. The classification depends on whether the cells are grown in a 2D-monolayer or a 3D-spheroid micro-environment. A metabolic-switch occurs, likely during the transition from anchorage-dependent to anchorage-independent growth. This represents a metabolic shift from a glycolytic to a more oxidative mitochondrial phenotype. More specifically, in 2D-monolayer cultures, and as discussed in more detail below, 100 nM DPI increased the number M-H cells by ˜7.5-fold over a 5-day period. In contrast, DPI, at exactly the same concentration, almost completely inhibited 3D-mammosphere formation, resulting in a population of anchorage-independent single live cells that were ˜60% depleted of S-H cells. Therefore, the same mitochondrial OXPHOS inhibitor (DPI) had completely opposite effects, depending on the 2D vs. 3D micro-environment of the e-CSCs. These results experimentally imply that M-H cell propagation in 2D-monolayers is driven by glycolysis, while the propagation of S-H cells in 3D-spheroids is driven by mitochondrial OXPHOS. Importantly, this suggests that a critical metabolic-switch is occurring, between the M-H and S-H CSC phenotypes, specifically altering their metabolic requirements.
[0039] This 2D-to-3D transition, or “epithelial-mesenchymal-transition (EMT)” is thought to be a more mesenchymal phenotype. In support of this notion. ALDH activity was progressively increased and was at its highest levels in e-CSCs derived from the 3D-spheroids, nearly 9-fold increased, directly supporting the assertions of the present approach. Importantly, ALDH activity is an established functional biomarker of the EMT and “boosts” the production of energy-rich NAD(P)H.
[0040] The identification of this unique, energy-driven, cancer cell sub-population will undoubtedly provide new opportunities for i) bio-banking and ii) new drug screening, as well as iii) the identification of novel metabolic targets, for the prevention of tumor recurrence and inhibiting the spread of metastatic disease.
[0041] Two human breast cancer cell lines. MCF7 and MDA-MB-468, were used as model systems, to dissect the role of metabolic heterogeneity in tumorigenesis. Results with MCF7 cells are shown in
TABLE-US-00001 TABLE 1 MCF7-derived e-CSCs cells demonstrate increased cell cycle progression. 2D-Monolayers (M) 3D-Spheroids (S) CC-Phase(%) M-L M-H S-L S-H G0/G1 81.25 53.23 61.50 37.32 S-phase 3.92 11.43 6.72 10.60 G2/M 8.53 21.23 11.72 32.43 Polyploid 3.71 10.74 9.03 17.13
[0042] Table 2, below, shows cell cycle data for cell populations from MDA-MB-468 e-CSCs. As with the MCF7 e-CSCs, these also cells demonstrate increased cell cycle progression. Table 3, also below, shows ALDH activity changes. The averages shown in both Tables 2 and 3 are from at least 3 independent experiments.
TABLE-US-00002 TABLE 2 MDA-MB-468 e-CSCs demonstrate increased cell cycle progression. 2D-Monolayers (M) 3D-Spheroids (S) CC-Phase (%) M-L M-H S-L S-H G0/G1 78.95 51.20 64.05 34.75 S-phase 2.96 12.18 9.03 18.10 G2/M 7.65 23.73 13.35 32.89 Polyploid 5.30 9.93 7.47 12.24
TABLE-US-00003 TABLE 3 MCF7-derived e-CSCs have increased ALDH activity. 2D-Monolayers (M) 3D-Spheroids (S) M-L M-H S-L S-H 0.52% 1.03% (1.98x) 2.13% (4.09x) 4.59% (8.83x)
[0043] The next series of analyses determined whether mitochondria may function as the metabolic “engines” to drive cellular hyper-proliferation in CSCs and, ultimately, anchorage-independent growth, leading to tumor recurrence and metastasis. The analysis also investigated whether two or more sub-populations of CSCs exist, depending on whether the cells are grown as 2D-monolayers or as 3D-spheroids). Cell auto-fluorescence was used as an endogenous marker of cellular energy metabolism, which directly reflects cellular content of flavin-containing compounds (FAD, FMN and riboflavin (Vitamin B2)), which are all high-energy cell metabolites.
[0044]
[0045] With respect to the data described herein, 2D-monolayers and 3D-spheroids were first collected and used to prepare single-cell suspensions. These suspensions were then subjected to flow-cytometry to isolate cells based on their auto-fluorescent properties. Briefly, the “Low-(L)” and “High-(H)” auto-fluorescent cell sub-populations were selected by gating, within the auto-fluorescence signal. Only cells with the least (bottom 5%) or the most (top 5%) auto-fluorescent signal were collected. Both the “Low” and “High” sub-populations of auto-fluorescent cells, generated from either 2D-monolayers (M-L vs. M-H) or 3D-spheroids (S-L vs. S-H) were then subjected to a detailed phenotypic characterization and separation. The M-H (“monolayer-high”) and S-H (“spheroid-high”) cell sub-populations were predicted to be the most energetic, based on their high (H) flavin-content.
[0046]
[0047] The next several paragraphs describe the characterization of the e-CSC phenotype, particularly with respect to proliferation, stemness, and bioenergetics. The e-CSC capacity for cell proliferation was assessed via cell cycle progression analysis. Representative cell cycle profiles (for different cell sub-populations of MCF7 are shown in
[0048] The M-H cell and S-H cell sub-populations were exceedingly hyper-proliferative, with a reduction of cells in the G0/G1-phase and dramatic increases in both the S-phase and the G2/M-phase. Also, the number of polyploid cells (DNA>2N) was increased considerably in both the M-H and S-H populations. Overall. S-H cells were the most hyper-proliferative, with >40% of the cells in S-phase and/or G2/M, and <40% of the cells in the G0/G1-phase of the cell cycle. S-H cells also had the largest number of polyploid cells, reaching approximately 12-17%, probably due to mitotic catastrophe. In contrast, M-L cells had the highest number of cells in the G0/G1-phase of the cell cycle (˜80%) and the lowest number of polyploid cells (˜3-5%). Also, M-L cells showed the lowest number of cells in S-phase (˜3-4%). These hyper-proliferative results with MCF7 cells (see Table 1, above) and MDA-MB-468 cells (see Table 2, above) arm also consistent with a high-energy phenotype. Given this phenotype, the M-H and S-H cells were designated as “energetic” CSCs, also referred to as e-CSCs in this disclosure.
[0049] The stem cell characteristics of the M-H and S-H cells were then assessed. Specifically, ALDH was used as a marker of “stemness” activity to carefully monitor the progressive enrichment of CSCs. The results are shown in
[0050] The mammosphere assay allows for the quantitative measurement of anchorage-independent growth, which is a functional read-out for “stemness” activity. High mammosphere formation in MCF7 cells directly correlates with high-flavin content. For example, M-H cells (from 2D-monolayers) and S-H cells (from 3D-spheroids) show the highest rates of mammosphere formation, as compared to the M-L and S-L sub-populations.
[0051] Mitochondrial mass was assessed using MitoTracker Deep Red vital staining. The mitochondrial mass in MCF7 cells correlates with high-flavin content. In particular. S-H cells (from 3D-spheroids) show that largest increases in mitochondrial mass, as seen by flow-cytometry with MitoTracker Deep Red vital staining.
[0052] Therefore, e-CSCs derived from 3D-spheroids were i) the most hyper-proliferative, ii) showed the largest increases in stemness characteristics (ALDH activity and anchorage-independent growth), and iii) had the highest mitochondrial mass. These phenotypic changes are highly suggestive of metabolic re-programming, especially towards more oxidative mitochondrial metabolism.
[0053] To characterize the bioenergetic phenotype of e-CSCs, the cell populations were subjected to metabolic flux analysis, using the Seahorse XFe96. Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were the measured properties. OCR results include basal respiration, maximal respiration, and ATP. ECAR results include glycolysis, glycolytic reserve, and glycolytic reserve capacity.
[0054] The data also demonstrates that e-CSCs have elevated levels of aerobic glycolysis. The extracellular acidification rate (ECAR) was measured, using the Seahorse XFe96 metabolic-flux analyzer.
[0055] In contrast, S-H cells demonstrated the highest increases in OCR, with a near 3-fold increase in basal respiration and a 4-fold increase in ATP production as seen in
[0056] The e-CSC phenotype allows for developing new therapeutics that target the metabolic nature of the sub-population. For example, it should be appreciated that e-CSCs may be targeted using OXPHOS inhibitors and/or CDK4/6 inhibitors. They may also be targeted with mitochondrial inhibitors. The following paragraphs describe examples of such therapeutic agents for targeting and eradicating e-CSCs, as an anti-cancer therapy. It should be appreciated that in some embodiments, these therapeutics may be used in conjunction with other anti-cancer therapies.
[0057] The first example therapeutic is DPI (Diphenyleneiodonium chloride), an OXPHOS inhibitor that specifically targets flavin-containing enzymes, especially those associated with FMN/FAD and mitochondrial complex I and II. DPI inhibits 3D-spheroid formation in MCF7 cells, and DPI selectively inhibits mitochondrial function without any toxic side effects. In prior studies. DPI did not induce changes in cell viability or apoptosis, but instead shifted the cells towards a more glycolytic phenotype.
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[0059] The S-H cells, on the other hand, are sensitive to DPI, as can be seen in
[0060] Complementary experiments were carried out with Ribociclib, a clinically-approved CDK4/6 inhibitor. Ribociclib is normally used to treat female breast cancer patients, in combination with letrozole (an aromatase inhibitor). Ribociclib was first developed by Astex Pharmaceuticals (Cambridge, UK) and Novartis. In 2017, Ribociclib was approved by the FDA and the European Medicines Agency, for the treatment of HR-positive, HER2-negative advanced or metastatic breast cancers. The drug's most common side-effects are: neutropenia, anemia and GI-distress. The data for treatment with Ribociclib in
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It should be appreciated that Ribocielib is one example of a CDK4/6 inhibitor, and that other CDK4/6 inhibitors may be used under the present approach. Other non-limiting examples of CDK4/6 inhibitors include, but are not limited to, abemacielib (Verzenio) and palbociclib (Ibrance).
[0061] Mitochondrial inhibitors may be used to eradicate e-CSCs. In the present approach, the mitochondrial inhibitors may be or include one or more of: a mitoribosein, the combination of an oxidative metabolism inhibitor and a glycolytic metabolism inhibitor, a repurposein, an antimitosein, a mitoketosein, a mitoflavosein, a mitoflavin, a TPP-derivative, an MDIVI-1 derivative, chloramphenicol, puromycin and other inhibitors of protein synthesis (including. e.g., aminoglycosides and rapamycin analogues), anti-parasitic drugs (such as, e.g., pyrvinium pamoate, and niclosamide), chloroquine, stiripentol, caffeic acid phenyl ester (CAPE), Vitamin C, 2-Deoxy-Glucose (2-DG), MCT1 inhibitors (AZD3965 and AR-C155858), D-Glucosamine, quercetin, and carvedilol. It should be appreciated that a therapeutic compound may fall under more than one category. The following paragraphs describe certain categories of mitochondrial biogenesis inhibitor therapeutics. For brevity, the related co-pending applications are incorporated by reference as if fully set forth herein.
[0062] A first category of therapeutics are mitoriboseins, as described in International Application No. PCT/US2018/022403, filed Mar. 14, 2018, and incorporated by reference in its entirety. The incorporated reference includes data for select mitoribosein compounds. Generally, mitoriboseins are mitochondrial inhibitor compounds that have anti-cancer and often antimicrobial activity, chemotherapy-sensitizing, radiosensitizing, and photosensitizing effects, as well as anti-aging effects. These compounds hind to either the large sub-unit or the small sub-unit of the mitoribosome (or in some instances, both) and inhibit mitochondrial biogenesis. Examples of mitoribosein groups, along with generic chemical structures and specific compounds, are described in the incorporated application, and include mitoribocyclines, mitoribomyeins, mitoribosporins, and mitoribofloxins.
[0063] A second category of mitochondrial biogenesis inhibitor therapeutics include combination therapies involving oxidative metabolism inhibitors and glycolytic metabolism inhibitors, e-CSCs in a mass may be reduced targeted and eradicated by administering a pharmaceutically effective amount of at least one oxidative metabolism inhibitor, and at least one glycolytic metabolism inhibitor. Inhibitors of oxidative metabolism may include members of the tetracycline family and the erythromycin family. Members of the tetracycline family include tetracycline, doxycycline, tigecycline, minocycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, rolitetracycline, chlortetracycline, omadacycline, and sarecycline. Members of the erythromycin family include erythromycin, azithromycin, and clarithromycin. Glycolytic metabolism inhibitors may be selected from inhibitors of glycolysis, inhibitors of OXPHOS, and inhibitors of autophagy. Examples of glycolysis inhibitors include 2-deoxy-glucose, ascorbic acid, and stiripentol, OXPHOS inhibitors include atoravaquone, irinotecan, sorafenib, niclosamide, and berberine chloride. Autophagy inhibitors include chloroquine. Data and further examples are described in International Application No. PCT/US2018/28587, filed Apr. 20, 2018, which is incorporated by reference in its entirety.
[0064] Some embodiments of combination therapies may take the form of a triple combination. For example, in some embodiments of the present approach, a first antibiotic inhibiting the large mitochondrial ribosome (such as, for example, members of the erythromycin family), and a second antibiotic inhibiting the small mitochondrial ribosome (such as, for example, members of the tetracycline family), may be administered with a pro-oxidant or an agent inducing mitochondrial oxidative stress % (e.g., low concentrations of Vitamin C, radiation therapy, among other examples). As a specific example, FDA-approved antibiotics doxycycline and azithromycin may be used in connection with one or more common dietary supplements (e.g., Vitamin C). In an example embodiment, treatment with a combination of doxycycline (at 1 μM), azithromycin (at 1 μM), and Vitamin C (at 250 μM) may be used as the mitochondrial biogenesis inhibitor. The pro-oxidant may be, in some embodiments, a therapeutic agent having a pro-oxidant effect. For example, the pro-oxidant may be a therapeutic agent at a concentration that causes the therapeutic agent to act as a reducing agent. U.S. Provisional Patent Application 62/780,488, filed Dec. 17, 2018 and incorporated by reference in its entirety, provides further description of triple combination therapies.
[0065] Antimitoseins are a third category of mitochondrial biogenesis inhibitors, described more fully in International Patent Application PCT/US2018/033466, filed May 18, 2018 and incorporated by reference in its entirety. Existing antibiotics having intrinsic anti-mitochondrial properties can be chemically modified to target the mitochondria and inhibit mitochondrial biogenesis. The term “antimitosein” broadly refers to an antibiotic having intrinsic anti-mitochondrial properties that is chemically modified to target the antibiotic to mitochondria. Previously, intrinsic anti-mitochondrial activity in antibiotics was considered to be an unwanted side-effect. Indeed, some potential antibiotics have been excluded from trials due to excessive anti-mitochondrial properties, and researchers have viewed anti-mitochondrial activity as a potential drawback. However, under the present approach, an antibiotic's intrinsic anti-mitochondrial activity can become the basic for an entirely new therapeutic. The antimitosein may be an antibiotic having intrinsic anti-mitochondrial properties chemically modified with a mitochondrial targeting signal (e.g., a chemical moiety). Chemical modification may be, for example, through covalent or non-covalent bonds. In some embodiments, the antibiotic is one of a member of the tetracycline family, the erthyromyein family, chloramphenicol, pyrvinium pamoate, atovaquone, and bedaquiline. The mitochondria-targeting signal may be at least one compound or moiety selected from the group comprising a membrane targeting signal and a mitochondrial ribosome-targeting signal. Examples of membrane targeting signals include short-chain (e.g., fewer than 6 carbon atoms in the chain) fatty acids and medium-chain (e.g., 6-12 carbon atoms in the chain) fatty acids, palmitic acid, stearic acid, myristic acid, and oleic acid. Examples of mitochondrial ribosome-targeting signals include tri-phenyl-phosphonium (TPP) and guanidinium-based moieties. TPP and guanidinium are non-toxic chemical moieties that functionally behave as a mitochondrial targeting signal (MTS) in living cells. Either may be bonded to an antibiotic, often through the use of a carbon spacer-arm or linking chain.
[0066] A fourth category of mitochondrial biogenesis inhibitors are mitoketoseins, non-carcinogenic compounds that bind to at least one of ACAT1/2 and OXCT1/2 and inhibit mitochondrial ATP production. These compounds are described more fully in International Application PCT/US2018/039354, filed Jun. 25, 2018, and incorporated by reference in its entirety. Generally, a mitoketosein targets the mitochondrial enzymes responsible for ketone re-utilization and that have anti-cancer and antibiotic properties. These compounds bind to either or both active catalytic sites of OXCT1/2 and ACAT1/2 to inhibit mitochondrial function.
[0067] Mitoflavoseins and mitoflavins are a fifth category of mitochondrial biogenesis inhibitors that may be used under the present approach. These compounds are described more fully in International Patent Application PCT/US2018/057093, filed Oct. 23, 2018 and incorporated by reference in its entirety. Mitoflavoseins are compounds that bind to flavin-containing enzymes and inhibit mitochondrial ATP production. Diphenyleneiodonium chloride (DPI) is an example of a mitoflavosein. It should be appreciated that a mitoflavosein may be modified with a mitochondrial targeting signal, such as discussed above with respect to antimitoseins. Mitoflavins, derivatives of riboflavin that inhibit mitochondrial function, may also be chemically modified with a mitochondrial targeting signal. For example, roseoflavin [8-Demethyl-8-(dimethylamino)-riboflavin or 8-Dimethylaminoriboflavin] is a naturally occurring anti-bacterial compound that is a derivative of riboflavin, which can be chemically modified to optimize its potential for targeting CSCs and inhibiting mitochondrial biogenesis. Lumichrome (7,8-Dimethylalloxazine) is a fluorescent photoproduct of riboflavin degradation, which also can be chemically modified to optimize its potential for targeting CSCs. Other common derivatives of riboflavin include: Alloxazine, Lumiflavine, 1,5-dihydroriboflavin and 1,5-dihydroflavin. Each of these riboflavin derivatives may be chemically modified with a mitochondrial targeting signal to form a mitoflavin, and may be used as a mitochondrial biogenesis inhibitor according to the present approach.
[0068] A sixth category of mitochondrial biogenesis inhibitors is TPP-derivative compounds that show not only a strong preference for uptake in cancer cells (bulk cancer cells, cancer stem cells, and energetic cancer stem cells), but also disrupt mitochondrial biogenesis in these cells. These TPP-derivative compounds are described more fully in International Patent Application PCT/US2018/062174, filed Nov. 21, 2018 and incorporated by reference in its entirety. As used with respect to TPP-derivatives, a derivative as known in the art is a compound that can be synthesized from a parent compound by replacing an atom with another atom or group of atoms. For example, a derivative of TPP is 2-butene-1,4-bis-TPP, which includes two phosphonium groups joined by butene. A derivative of 2-butene-1,4-bis-TPP, then, could include replacement of one or more phenyl groups with another compound, such as a halogen or an organic compound. For the sake of brevity, this disclosure does not identify all of the potential derivatives, as the description should be adequate for a person of ordinary skill in the art. Other examples of TPP-derivative compounds that may be used as mitochondrial biogenesis inhibitors according to the present approach include 2-butene-1,4-bis-TPP; derivatives of 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; derivatives of 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; derivatives of 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; derivatives of 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; derivatives of 1-naphthylmethyl-TPP; p-xylylenebis-TPP; and derivatives of p-xylylenebis-TPP. Of course, it should be appreciated that the foregoing list is not an exhaustive list of TPP-derivatives.
[0069] Repurposeins are a seventh category of mitochondrial biogenesis inhibitors that may be used in embodiments of the present approach. International Patent Application PCT/US2018/062956, filed Nov. 29, 2018 and incorporated by reference in its entirety, describes these compounds more fully. Generally, “repurposeins” are compounds having intrinsic anti-mitochondrial properties that are chemically modified to target the compounds to mitochondria. Such compounds may include, for example, FDA-approved pharmaceuticals, nutraceuticals, and supplements, among others. Compounds having intrinsic anti-mitochondrial properties may be chemically modified with one or more mitochondrial targeting signals as described above. Examples of compounds having intrinsic anti-mitochondrial properties include berberine chloride, quercetin, niclosamide, acriflavinium hydrochloride, sorafenib, emetine dihydrochloride, dactinomycin, plicamycin, suloctidil, teniposide, pentamidine isethionate, daunorubicin, thioguanine, amsacrine, phenformin hydrochloride, irinotecan hydrochloride, mitomycin, hydroxyprogesterone caproate, cyclosporine, lanatoside c, mercaptopurine, quinacrine hydrochloride, and fenofibrate. In some embodiments, the compound may be one or more of neomycin, puromycin, rapamycin (and its derivatives, such as everolimus), G418, trovafloxacin, levonoxacin, avocatin B, clarithomycin, ciprofloxacin, spiramycin, telithromycin, norfloxacin, moxifloxacin, ofloxacin, minocycline, tetracycline, demethylchlortetracycline, a member of the tetracycline family, a member the erythromycin family, clindamycin, metronidazole, linezolid, mupirocin, vancomycin, clindamycin, cephalosporin, ciprofloxacin, streptomycin, amoxicillin, and azelaic acid. It should be noted that a repurposein formed from an antibiotic may also be referred to as an antimitosein.
[0070] An eighth category of mitochondrial biogenesis inhibitors that may be used in the present approach is MDIVI-1 derivatives, as described in International Patent Application PCT/US2018/066247, filed Dec. 18, 2018 and incorporated by reference in its entirety. Mitochondrial division inhibitor-1 (mDIVI-1) is a small molecule that selectively and reversibly inhibits DRP1. MDIVI-1 has been shown to target DRP1 by binding and suppressing both the DRP1 self-assembly into ring-like structures around the mitochondria and its capacity to catalyze GTP hydrolysis. The present approach may take the form of a mitochondrial fission inhibitor 1 (mDIVI-1) derivative having the general formula:
##STR00003##
or a pharmaceutically acceptable salt thereof, wherein each of R1 through R8 may be selected from the group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivatives, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-bused derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arenes, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, benzoic acid-based derivatives, and a mitochondrial targeting signal. In some embodiments, at least one R-group is a mitochondrial targeting signal, such as palmitic acid, stearic acid, myristic acid, and oleic acid, a short-chain fatty acid, a medium-chain fatty acid, tri-phenyl-phosphonium (TPP), a TPP-derivative, a lipophilic cation, and 10-N-nonyl acridine orange. In some embodiments, at least one R-group is a mitochondrial targeting signal, such as one of 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; I-naphthylmethyl-TPP; p-xylylenebis-TPP; a derivative of 2-butene-1,4-bis-TPP; a derivative of 2-chlorobenzyl-TPP; a derivative of 3-methylbenzyl-TPP; a derivative of 2,4-dichlorobenzyl-TPP; a derivative of 1-naphthylmethyl-TPP; and a derivative of p-xylylenebis-TPP. It should be appreciated that MDIVI-1 derivatives may be used us mitochondrial inhibitors under the present approach, with one or more of the chemical modifications described in this paragraph.
[0071] Although the data described above relates to breast cancer cells, it should be appreciated that the e-CSC phenotype is not limited to MCF7 cells. Data for MDA-MB-468 cell subpopulations shows that e-CSCs have common characteristics across different cancer types.
[0072]
[0073]
[0074] The following paragraphs describe the proteomics analysis of e-CSCs derived from MCF7 3D-spheroid cells. Label-free unbiased proteomics analysis was used to describe the mechanistic basis for the biogenesis of e-CSCs. As a consequence, 225 proteins were identified that were significantly up-regulated by ≥1.5-fold. Conversely, 187 proteins were significantly down-regulated. For simplicity, the analysis focused on the specific protein products that were up-regulated and these are shown in Table 4, below. Interestingly, 48 of these proteins (representing ˜20% of the total number) were specifically related to mitochondrial energy production and/or mitochondrial biogenesis. These 48 proteins are identified in Table 5, below. This is consistent with the functional observations described above, that e-CSCs demonstrate a near 4-fold increase in both mitochondrial mass and mitochondrial ATP production.
[0075] Table 6 shows further bioinformatics analysis, assembling the proteins into distinct functional groups. These functional classes include senescence, the anti-oxidant response, “stemness,” cytoskeletal proteins (suggestive of an EMT), glutamine metabolism, NADH/NADPH synthesis, flavin-containing enzymes, autophagy/lysosomes, peroxisomes, and various cellular markers (epithelial, cell surface, S100 family proteins, RABs, annexins, PARP, calcium signaling). Interestingly, CDKN1A (p21 WAF), which is a CDK-inhibitor and senescence marker, is highly up-regulated by 17.22-fold in e-CSCs. This finding is consistent with the idea that CSCs originate from senescent cells.
[0076] However, e-CSCs are hyper-proliferative, so they likely escaped from senescence. This may have occurred through the over-expression of anti-oxidant enzymes or the over-production of NADH/NADPH. Loss of glutaredoxin expression is known to be sufficient to induce a senescence phenotype in cells, in a p21-dependent manner. Therefore, the observed over-expression of glutaredoxin (by 10.79-fold) may be sufficient to actually overcome senescence, allowing the creation of e-CSCs.
[0077] Importantly, glutaredoxin expression is known to drive mitochondrial biogenesis by directly regulating the activation state of two key mitochondrial proteins, namely HSP60 and DJ-1 (Park7) (35). HSP60 is a mitochondrial chaperone, which facilitates the proper folding of newly synthesized or imported mitochondrial proteins, while DJ-1 functionally maintains the activity of mitochondrial complex 1 and SOD2. As a consequence, glutaredoxin expression specifically maintains the integrity of mitochondria and elevates ATP synthesis. Glutaredoxin's ability to regulate mitochondrial energy production is also linked to cell cycle progression. As such, glutaredoxin allows cells to successfully pass through the GUS transition, in a CDK4-dependent manner, thereby avoiding the cell-cycle arrest associated with senescence.
[0078] The 10.24-fold up-regulation of ALDH3A1 also provides significant anti-oxidant power, as ALDH isoforms are known to functionally increase the cellular levels of NADH/NADPH. Also, the main isoform up-regulated in e-CSCs, namely ALDH3A1, is known to be associated with tumorigenesis, metastasis and drug-resistance. Ingenuity Pathway Analysis (IPA) of the proteomics data sets referenced above confirmed the up-regulation of the anti-oxidant response and cell cycle progression in e-CSCs, as well as the changes in mitochondrial function IPA analysis of the proteomics data sets showed the activation of upstream regulator PPARGC-1-Alpha 1301, also known as PGC-1-Alpha, the major mitochondrial transcription factor. Also, a canonical pathway assessment identified the NRF2-mediated oxidative stress response pathway, and the cell cycle G2/M DNA damage checkpoint regulation pathway as significantly up-regulated.
[0079] In vivo data demonstrates that e-CSC proteins are transcriptionally up-regulated in human breast cancer patients. Pre-existing mRNA profiling data, obtained from the laser-capture analysis of N=28 human breast cancer patient tumor samples were used to determine if e-CSCs proteins were also transcriptionally up-regulated in human breast cancer cells in vivo. In this data set, breast cancer cells were physically separated from adjacent tumor stromal cells, using laser-captured mediated micro-dissection. The results of this intersection are presented in Table 7. The results indicate that out of the 225 proteins that were up-regulated in e-CSCs, nearly one-third of these gene products were transcriptionally up-regulated in human breast cancer cells (70/225=31.11%). In addition, many of these gene products were shared, including 20 mitochondrial related genes (20/70=28.57%). These results provide genetic evidence that demonstrates the clinical relevance of e-CSCs in the study of human breast cancers.
[0080] In connection with the prior work, a short anti-oxidant response signature in e-CSCs has been developed that predicts poor clinical outcome in breast cancer patients. The next paragraphs describe identifying subsets of e-CSCs proteins that have prognostic value in terms of predicting clinical outcome in human breast cancer patients. A well-defined set of high-risk ER(+) patients (luminal A) that received hormone-therapy (mostly-tamoxifen), with local Lymph-Node (LN) metastasis at diagnosis, as well as >150 months (12.5 years) of follow-up data were used for this assessment. In all of these cancer patients, their breast tumor tissues also underwent genomic transcriptional profiling.
[0081] Kaplan-Meier (K-M) analysis was used to specifically determine whether these e-CSCs proteins listed in Table 7 had prognostic value by determining their effects on the Hazard-Ratio (HR), by employing the Log-Rank test to determine statistical significance. Based on this analysis, a four-gene signature consisting of members of the anti-oxidant response and NAD(P)H metabolism, namely NQO1, ALDH5A1, TXNR and RRM2, was developed. Table 8, below, summarizes the results. The other 66 gene products tested did not show this prognostic ability.
[0082] The K-M curves shown in
[0083] These four gene products (NQO1, ALDHSA1, TXNR, and RRM2) were tested individually and all showed a >2-fold increase in the HR. In addition, when combined into a short signature, this resulted in a HR of nearly 4, with a p-value of 4.1e-05. As a consequence, under the present approach this anti-oxidant signature may be used to predict tumor recurrence (FRS) in patients receiving hormonal therapy.
[0084] Similarly, the inventors also recognized that the transcriptional elevation of 3 out of 4 of these gene products (ALDH5A1, TXNR and RRM2) was effectively able to predict distant metastasis (DMFS), with HRs of 2.86 to 3.64, and p-values of 0.003 to 0.00035. Table 9, below, shows these results. Thus, under the present approach a three-gene signature of ALDH5A1. TXNR and RRM2 may be used to identify a risk of distant metastasis.
[0085] It should be appreciated that the anti-oxidant response in e-CSCs allowed the inventors to successfully identify gene products with predictive value, for anticipating the onset of recurrence and/or metastasis, in breast cancer patients that ultimately underwent treatment failure, in response to hormonal therapy. K-M curves for larger groups of breast cancer patients are also shown
[0086] Embodiments of the present approach may involve measuring or determining the expression level of one or more genes in the e-CSC gene signature (NQO1, ALH5A1, TXNR, and RRM2). The e-CSC gene signature has prognostic value with respect to the presence of e-CSCs, tumor recurrence due tat least in part) to e-CSCs, and distant metastasis due (at least in part) to e-CSCs. Up-regulation of one or more of the genes in the e-CSC gene signature may be used as a biomarker indicating that treatment for e-CSCs may be beneficial. Treatment for e-CSCs includes administering a pharmaceutically effective amount of at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor. Additionally, e-CSCs may be treated with at least one mitochondrial biogenesis inhibitor. The treatment for e-CSCs may reduce or eliminate the likelihood of distant metastasis and/or tumor recurrence, and also may improve the effectiveness of other cancer therapies. It should be appreciated that gene expression levels may be measured using assays known to those having ordinary skill in the art. Gene expression may be measured based on the protein gene product, and common techniques include expression proteomics. Western blotting, and enzyme-linked immunosorbent assay (sometimes referred to as the ELISA assay). Gene expression may also be measured based on mRNA levels, and common techniques for mRNA level measurement include Northern blotting and reverse transcription then quantitative polymerase chain reaction (also called RT-qPCR). The threshold or baseline level(s) may be obtained from available literature and/or databases known in the art. Also, the threshold or baseline level(s) may be obtained from using an assay on a biologic sample representing a normal, healthy cell line. As those having at least an ordinary level of skill in the art will appreciate, the threshold or baseline level(s) may also be determined from in vivo data of patients having the same cancer, but no symptoms of tumor recurrence and/or distant metastasis. For example, in some embodiments the threshold data may be derived from the K-M-plotter source referenced above. In such embodiments, overexpression of one or more genes from the e-CSC gene signature in a mass, relative to the threshold data, indicates the presence of e-CSCs, and is prognostic of a likelihood of tumor recurrence and/or metastasis. The overexpression may be quantified as a ratio, and the ratio for determining overexpression depends on the embodiment. For example, in some embodiments overexpression may be determined if the quotient of the determined level divided by the threshold level is greater than 1.2. As further examples, in some embodiments the ratio may be 1.4, and in some embodiments is may be 1.6. It should be appreciated that the present approach is not limited to a particular threshold or metric for indicating overexpression 13 the person of ordinary skill in the art may identify a threshold data source for a particular cancer, and select the ratio for diagnosing overexpression of one or more genes of the e-CSC gene signature. This gene signature has prognostic value, as discussed above, and may be used as a biomarker of e-CSCs and the risk of tumor recurrence and/or metastasis due (at least in part) to e-CSCs. In turn, the e-CSC gene signature may be used to identify instances in which a pharmaceutically effective amount of at least one of an OXPHOS inhibitor, a CDK4/6 inhibitor, and/or a mitochondrial biogenesis inhibitor, may be administered to target e-CSCs in a mass, treat (i.e., reduce the likelihood of) distant metastasis and/or tumor recurrence, and treat cancer.
[0087] One explanation for the prognostic value of this compact gene signature is that an adaptive anti-oxidant response drives resistance to both chemotherapy and radiotherapy in cancer patients. In addition, TXNR and RRM2 both are key enzymes that provide the required precursors for nucleotide biosynthesis and, hence, cell cycle progression. The anti-oxidant response signatures of the present approach may also be useful for identifying breast cancer patients that could benefit from treatment with i) mitochondrial inhibitors or ii) CDK inhibitors, especially in the context of preventing tumor recurrence and/or distant metastasis. In some embodiments, these treatments may be used individually, or in combination with other therapies, such as (but not limited to) chemotherapy and radiation therapy. Thus, in the future, ROS production in e-CSCs, under both 2D and 3D microenvironmental conditions, will be used to validate that ROS production is driving this anti-oxidant response signature and contributes to their overall energetic phenotype.
[0088] The following paragraphs describe the methodologies and materials used in connection with the foregoing. It should be appreciated by those of ordinary skill in the art that variations may be made without departing from the present approach. With respect to the breast cancer cell models and other reagents, human breast cancer cell lines, MCF7 (ER(+)) and MDA-MB-468 (triple-negative), were obtained commercially from the ATCC. Both cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM: GIBCO), supplemented with 10% FBS, 1% Glutamax and 1% Penicillin-Streptomycin. All cell lines were maintained at 37° C. in 5% CO2. DPI and Ribociclib were purchased from Sigma-Aldrich, Inc.
[0089] Cell sorting: Flow-cytometry and collection of auto-fluorescent cells: MCF7 and MDA-MB-468 cells were first grown either as a 2D-monolayer or as 3D-spheroids. Then, they were collected and dissociated into a single-cell suspension, prior to analysis or sorting by flow-cytometry with the SONY SH800 Cell Sorter. Briefly, auto-fluorescent cells were excited with a 488 nm blue laser and selected at the intersection with filters 525/50 and 585/30. The “Low” and “High” auto-fluorescent cell sub-populations were selected by gating, within the auto-fluorescence signal. Only cells with the least (bottom 5%) or the most (top 5%) auto-fluorescence signal were collected. The cells outside the gates were discarded during sorting, due to the gate settings. However, such settings am required, to ensure high-purity during sorting. To better characterize the auto-fluorescent cell sub-populations, the following flow-cytometry markers were used: ALDEFLUOR-assay (StemCell technologies, Durham, N.C., USA); and MitoTracker Deep Red (Thermo Fisher Scientific). Hoescht (Thermo Fisher Scientific) was used for cell cycle analysis. Data were analyzed with FlowJo 10.1 software.
[0090] Preparing cells for auto-fluorescent cell sorting by flow-cytometry: The following protocol was used to acquire and sort auto-fluorescent cells from 2D-monolayers or 3D-spheroid cell suspensions. For 2D-monolayers. MCF7 and MDA-MB-468 were seeded in a 225 cm2 flask and when ˜70% confluence was reached, 5 ml of 0.025% trypsin was added to the flasks and incubated at 37° C. for 5 minutes. After that the cells were re-suspended in media and centrifuged at 300 g for 5 min. After centrifugation, the cell pellets were adjusted to a concentration of 106 cells/ml in in PBS Ca/Mg for acquisition or in sorting buffer (1×PBS containing 3% (v/v) FBS and 2 mM EDTA) for FACS.
[0091] For 3D-spheroid suspensions, after 5 days of growth under low-attachment condition, the spheres were collected from six 225 cm2 flasks pre-coated with poly-HEMA and gently centrifuged at 100 g for 5 min. After centrifugation, 1 ml of 0.025% of trypsin was added to the “sphere-pellet” and incubated them at 37° C. for 5 minutes. Using a 25-gauge needle, the sphere-suspension was passed through the syringe 4 times. The sphere suspension was then centrifuged again at 100 g for 5 min, and the sphere-pellet was re-suspended in i) PBS Ca/Mg for acquisition or ii) in sorting buffer (1×PBS containing 3% (v/v) FBS and 2 mM EDTA) for FACS and the suspension was “syringed” again 4 times. After creating these single-cell suspensions, they were subjected to standard flow-cytometry (using the SONY SH800 Cell Sorter) to isolate the auto-fluorescent cell sub-populations, as indicated above. Examples of flow-cytometry plots are included in the figures, and the gating strategy is shown.
[0092] Mammosphere formation assay (for generating 3D-spheroids): A single-cell suspension was prepared using enzymatic, and manual disaggregation (25 gauge needle). Then, cells were plated at a density of 500 cells/cm2 in mammosphere medium (DMEM-F12+B27+20 ng/ml EGF+PenStrep) under non-adherent conditions, in culture dishes pre-coated with (2-hydroxyethylmethacrylate) (poly-HEMA, Sigma, #P3932), called “mammosphere plates.” Cells were grown for 5 days and maintained in a humidified incubator at 37° C. After 5 days of culture, 3D-spheres>50 μm were counted using an eye piece (“graticule”), and the percentage of cells plated which formed spheres was calculated and is referred to as percent mammosphere formation, and was normalized to one (1=100% MSF). Mammosphere formation efficiency was analyzed in both the “low” and “high” sub-populations of auto-fluorescent cells, generated from either 2D-monolayers (M-L vs. M-H) or 3D-spheroids (S-L vs. S-H). All mammosphere experiments were performed in triplicate, at least 3 times independently.
[0093] ALDEFLUOR assay: The level of ALDH activity was assessed, by using the fluorescent reagent ALDEFLUOR. The ALDEFLUOR kit (StemCell technologies, Durham, N.C., USA) was used to detect the cell sub-populations with various amounts of ALDH enzymatic activity by FACS (Attune N×T Flow Cytometer). Briefly, 1×105 were incubated in 1 ml ALDEFLUOR assay buffer containing ALDH substrate (5 μl/ml) for 40 minutes at 37° C. In each experiment a sample of cells was stained under identical conditions with 30 μM of diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor, as a negative control The ALDH-positive population was established, according to the manufacturer's instructions and was evaluated using 50,000 cells. All the ALDH experiments were performed three times independently.
[0094] Seahorse XFe96 metabolic flux analysis: Real-time oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) rates were determined using the Seahorse Extracellular Flux (XFe96) analyzer (Seahorse Bioscience. USA). Briefly, 2×104 cells per well were seeded into XFe96 well cell culture plates after sorting, and incubated for 12 h to allow cell attachment. After 12 hours of incubation, cells were washed in pre-warmed XF assay media (or for OCR measurement. XF assay media supplemented with 10 mM glucose, 1 mM Pyruvate, 2 mM L-glutamine and adjusted at 7.4 pH). Cells were then maintained in 175 μL/well of XF assay media at 37° C. in a non-CO2 incubator for 1 hour. During the incubation time, 25 μL of 80 mM glucose, 9 μM oligomycin, and 1M 2-deoxyglucose (for ECAR measurement) or 10 μM oligomycin, 9 μM FCCP, 10 μM rotenone, 10 μM antimycin A (for OCR measurement), was loaded in XF assay media into the injection ports in the XFe96 sensor cartridge (20, 21). Measurements were normalized by protein content (SRB assay) and Hoechst 33342 content. Data sets were analyzed using XFe96 software and GraphPad Prism software, using one-way ANOVA and Student's t-test calculations. All experiments were performed in quintuplicate, three times independently.
[0095] Vital mitochondrial staining: Cells were trypsinized and re-suspended into a 1×106 cell/ml solution in PBS, 10 nM of MitoTracker Deep-Red (Thermo Fisher Scientific) was added for 30 minutes at 37° C. before centrifugation and re-suspension in PBS Ca/Mg for FACS analysis (ATTUNE N×T) or Cell Sorting (SONY SH 800). All subsequent steps were performed in the dark. Data analysis was performed using FlowJo software.
[0096] Cell cycle analysis: The cell-cycle analysis was performed on the auto-fluorescent cell sub-populations, by FACS analysis using the SONY Cell Sorter. Briefly, after trypsinization, the re-suspended cells were incubated with 10 ng/ml of Hoescht solution (Thermo Fisher Scientific) for 40 minutes at 37° C. under dark conditions. Following a 40 minute period, the cells were washed and re-suspended in PBS Ca/Mg for acquisition or in sorting buffer (1×PBS containing 3% (v/v) FBS and 2 mM EDTA) for FACS. We analyzed 50,000 events per condition. Gated cells were manually-categorized into cell-cycle stages.
[0097] Statistical analysis: All analyses were performed with GraphPad Prism 6. Data were represented as mean±SD (or ±SEM where indicated). All experiments were conducted at least 3 times independently, with >3 technical replicates for each experimental condition tested (unless stated otherwise, e.g., when representative data is shown). Statistically significant differences were determined using the Student's t test or the analysis of variance (ANOVA) test. For the comparison among multiple groups, one-way ANOVA were used to determine statistical significance. P<0.05 was considered significant and all statistical tests were two-sided. In the drawings, * indicatesP<0.05; ** indicates P<0.005; and *** indicates P<0.0005.
[0098] Proteomics analysis: Label-free unbiased proteomics and Ingenuity pathway analysis (IPA) were carried out, essentially as previously described, using standard protocols, with relatively minor modifications. For IPA, unbiased interrogation and analysis of the proteomic data sets was carried out by employing the IPA bioinformatics platform (Ingenuity systems, http://www.ingenuity.com). IPA assists with data interpretation, via the grouping of differentially expressed genes or proteins into known functions and pathways. Pathways with a z score of >+2 were considered as significantly activated, while pathways with a z score of <−2 were considered as significantly inhibited.
[0099] Clinical relevance of e-CSC marker proteins: To validate the clinical relevance of our findings, the inventors first assessed whether the identified e-CSC targets in MCF7 cells were also transcriptionally upregulated in human breast cancer cells in vivo. For this purpose, the inventors employed a published clinical data set of N=28 breast cancer patients in which their tumor samples were subjected to laser-capture micro-dissection (26), to physically separate epithelial cancer cells from their adjacent tumor stroma.
[0100] Kaplan-Meier (K-M) analyses: To perform K-M analysis on mRNA transcripts, the inventors used an open-access online survival analysis tool to interrogate publically available microarray data from up to 3.455 breast cancer patients. This allowed us to determine their prognostic value (27). For this purpose, the inventors primarily analyzed data from ER(+) patients that were LN(+) at diagnosis and were of the luminal A sub-type, that were primarily treated with tamoxifen and not other chemotherapy (N=150 patients). In this group, 100% the patients received some form of hormonal therapy and ˜95% of them received tamoxifen. Biased and outlier array data were excluded from the analysis. This allowed us to identify metabolic gene transcripts, with significant prognostic value. Hazard-ratios were calculated, at the best auto-selected cut-off, and p-values were calculated using the log-rank test and plotted in R. K-M curves were also generated online using the K-M-plotter (as high-resolution TIFF files), using univariate analysis: http://kmplot.com/analysis/index.php?p=service&cancer=breast. This allowed us to directly perform in silico validation of these metabolic biomarker candidates. The 2017 version of the database was utilized for all these analyses, while virtually identical results were also obtained with the 2014 and 2012 versions.
[0101] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the approach. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising.” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
[0102] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the claims of the application rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
TABLE-US-00004 TABLE 4 Proteomic analysis of e-CSCs, derived from MCF7 3D-Spheroids Fold-Change Symbol Gene Description (Up-regulation) BCAS1 Breast carcinoma-amplified sequence 1 119.37 CDKN1A Cyclin-dependent kinase inhibitor 1 (p21-WAF/CDK-inhibitor) 17.22 GLRX Glutaredoxin-1 10.79 ALDH3A1 Aldehyde dehydrogenase, dimeric NADP-preferring 10.24 CEACAM6 Carcinoembryonic antigen-related cell adhesion molecule 6 9.66 CYP1A1 Cytochrome P450 1A1 6.60 ELMOD2 ELMO domain-containing protein 2 4.73 MAOA Amine oxidase [flavin-containing] A 4.73 KRT10 Keratin, type cytoskeletal 10 4.59 IGFBP2 Insulin-like growth factor-binding protein 2 4.20 QPRT Nicotinate-nucleotide pyrophosphorylase [carboxylating] 3.72 MVP Major vault protein 3.61 CEACAM5 Carcinoembryonic antigen-related cell adhesion molecule 5 3.38 CLU Clusterin 3.13 QSOX1 Sulfhydryl oxidase 1 2.93 CIB1 Calcium and integrin-binding protein 1 2.90 VGF Neurosecretory protein VGE 2.90 ANXA1 Annexin A1 2.87 AKR1C3 Aldo-keto reductase family 1 member C3 2.79 LAMA5 Laminin subunit alpha-5 2.72 CDC42BPG Serine/threonine-protein kinase MRCK gamma 2.69 RAB27B Ras-related protein Rab-27B 2.69 CHMP6 Charged multivesicular body protein 6 2.62 TUBA4A Tubulin alpha-4A chain 2.60 PARP4 Poly [ADP-ribose] polymerase 4 2.55 RAB27A Ras-related protein Rab-27A 2.54 EVPL Envoplakin 2.48 KLK11 Kallikrein- 11 2.46 MAOB Amine oxidase [flavin-containing] B 2.45 DPP7 Dipeptidyl peptidase 2 2.43 AKR1C2 Aldo-keto reductase family 1 member C2 2.41 SFXN3 Sideroflexin-3 2.40 MIC13 MICOS complex subunit MIC13
mitochondrial 2.36 GM2A Ganglioside GM2 activator 2.36 SCRN2 Secernin-2 2.34 SULT1A1 Sulfotransferasc 1A1 2.34 RRM2 Ribonucleoside-diphosphate reductase subunit M2 2.34 SERPINA3 Alpha-1-antichymotrypsin 2.33 SLC6A14 Sodium- and chloride-dependent neutral and basic amino acid transporter B(0+) 2.30 AGA N(4)-(beta-N-acetylglucosaminyl)-L-asparaginasc 2.30 SYTL2 Synaptotagmin-like protein 2 2.30 MPV17 Protein Mpv17 2.28 KIAA0319L Dyslexia-associated protein KIAA0319-like protein 2.25 B3GAT3 Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 3 2.22 PON2 Serum paraoxonase/arylesterase 2 2.22 OXSM 3-oxoacyl-[acyl-carrier-protein] synthase, mitochondrial 2.22 TOM1L2 TOM1-like protein 2 2.22 STOM Erythrocyte band 7 integral membrane protein 2.18 MROH1 Maestro heat-like repeat-containing protein family member 1 2.17 PI4K2A Phosphatidylinositol 4-kinase type 2-alpha 2.17 FECH Ferrochelatase, mitochondrial 2.16 MCU Calcium uniporter protein, mitochondrial 2.13 S100P Protein S100-P 2.11 RDH13 Retinol dehydrogenase 13 2.08 PPL Periplakin 2.08 TSPAN31 Tetraspanin-31 2.03 TIMP1 Metalloproteinase inhibitor 1 2.02 GCLC Glutamate-cysteine ligase catalytic subunit 2.01 NEBL Nebulette 2.01 MUC5B Mucin-5B 1.98 CTSH Cathepsin H 1.98 GNS N-acetylglucosamine-6-sulfatase 1.97 S100A10 Protein S100-A10 1.96 INPP4B Type II inositol 3,4-bisphosphate 4-phosphatase 1.96 PHYKPL 5-phosphohydroxy-L-lysine phospho-lyase 1.95 ASAH1 Acid ceramidase 1.94 DHRS1 Dehydrogenase/reductase SDR family member 1 1.93 PEX14 Peroxisomal membrane protein PEX14 1.91 PTGR1 Prostaglandin reductase 1.91 NQO2 Ribosyldihydronicotinamide dehydrogenase [quinone] 1.90 STARD3NL STARD3 N-terminal-like protein 1.88 MGST1 Microsomal glutathione S-transferase 1.88 CMC1 COX assembly mitochondrial protein homolog 1.87 DGAT1 Diacylglycerol O-acyltransferase 1 1.87 RAB24 Ras-related protein Rab-24 1.87 GDPD3 Lysophospholipase D GDPD3 1.86 DCLK1 Serine/threonine-protein kinase DCLK1 1.85 PSAP Prosaposin 1.85 MGST3 Microsomal glutathione S-transferasc 3 1.84 ANO10 Anoctamin-10 1.84 CASK Peripheral plasma membrane protein CASK 1.84 LGALS3BP Galectin-3-binding protein 1.83 GAA Lysosomal alpha-glucosidase 1.83 ISCU Iron-sulfur cluster assembly enzyme ISCU, mitochondrial 1.83 GALNS N-acetylgalactosamine-6-sulfatase 1.82 DECR2 Peroxisomal 2,4-dienoyl-CoA reductase 1.81 ABAT 4-aminobutyrate aminotransferase
mitochondrial 1.81 PALM3 Paralemmin-3 1.81 ABCB6 ATP-binding cassette sub-family B member 6, mitochondrial 1.80 GFER FAD-linked sulfhydryl oxidase ALR 1.80 CD59 CD59 glycoprotein 1.80 SLC39A11 Zinc transporter ZIP11 1.80 CAPN2 Calpain-2 catalytic subunit 1.79 FAM174B Membrane protein FAM174B 1.79 TMEM160 Transmembrane protein 160 1.79 ACADSB Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial 1.79 FAM8A1 Protein FAM8A1 1.79 CAPS Calcyphosin 1.79 ARMC10 Armadillo repeat-containing protein 10 1.78 TMTC3 Transmembrane and TPR repeat-containing protein 3 1.78 SCFD2 Sec1 family domain-containing protein 2 1.78 HDHD3 Haloacid dehalogenase-like hydrolase domain-containing protein 3 1.78 RETSAT All-trans-retinol 13,14-reductase 1.77 COQ9 Ubiquinone biosynthesis protein COQ9, mitochondrial 1.77 SPATA20 Spermatogenesis-associated protein 20 1.77 EML2 Echinoderm microtubule-associated protein-like 2 1.77 ALDH5A1 Succinate-semialdehyde dehydrogenase, mitochondrial 1.76 GRN Granulins 1.76 CPT2 Carnitine O-palmitoyltransferase 2, mitochondrial 1.76 PEX11B Peroxisomal membrane protein PEX11B 1.76 HMGCL Hydroxymethylglutaryl-CoA lyase, mitochondrial 1.75 GSTK1 Glutathione S-transferase kappa 1 1.75 DHRS7B Dehydrogenase/reductase SDR family member 7B 1.75 FDXR NADPH:adrenodoxin oxidoreductase, mitochondrial 1.75 EPS8L1 Epidermal growth factor receptor kinase substrate 8-like protein 1 1.74 SLC22A18 Solute carrier family 22 member 18 1.74 CYCS Cytochrome e 1.74 MAPRE3 Microtubule-associated protein RP/EB family member 3 1.74 SQOR Sulfide:quinone oxidoreductase, mitochondrial 1.73 PDIA5 Protein disulfide-isomerase A5 1.73 HIGD1C HIG1 domain family member 1C 1.72 EML3 Echinoderm microtubule-associated protein-like 3 1.72 PCLAE PCNA-associated factor 1.72 ATP6V0A1 V-type proton ATPase 116 kDa subunit a isoform 1 1.71 TAOK3 Serine/threonine-protein kinase TAO3 1.71 ITGAV Integrin alpha-V 1.71 CAMK2D Calcium/calmodulin-dependent protein kinase type II subunit delta 1.70 SLC9A1 Sodium/hydrogen exchanger 1 1.69 CALML5 Calmodulin-like protein 5 1.69 HMOX1 Heme oxygenase 1 1.69 RNASET2 Ribonuclease T2 1.69 SELENBP1 Methanethiol oxidase 1.68 ACAA1 3-ketoacyl-CoA thiolase, peroxisomal 1.68 FKBP11 Peptidyl-prolyl cis-trans isomerase FKBP11 1.68 RRM2B Ribonucleoside-diphosphate reductase subunit M2 B 1.68 MLYCD Malonyl-CoA decarboxylase, mitochondrial 1.67 ENDOG Endonuclease G, mitochondrial 1.67 HPDL 4-hydroxyphenylpyruvate dioxygenase-like protein 1.67 CYB5R1 NADH-cytochrome b5 reductase 1 1.66 KIF1A Kinesin-like protein KIF1A 1.66 ENTPD8 Ectonucicoside triphosphate diphosphohydrolase 8 1.66 DLGAP4 Disks large-associated protein 4 1.66 IVD Isovaleryl-CoA dehydrogenase, mitochondrial 1.66 MRPS18C 28S ribosomal protein S18c, mitochondrial 1.66 CTSD Cathepsin D 1.66 HIBCH 3-hydroxyisobutyryl-CoA hydrolase, mitochondrial 1.66 HS1BP3 HCLS1-binding protein 3 1.66 MISP Mitotic interactor and substrate of PLK1 1.66 ANXA2 Annexin A2 1.65 CD44 CD44 antigen 1.65 MSRB2 Methionine-R-sulfoxide reductase B2, mitochondrial 1.65 GLB1 Beta-galactosidase 1.64 CPD Carboxypeptidase D 1.64 TACSTD2 Tumor-associated calcium signal transducer 2 1.64 COMTD1 Catechol O-methyltransferase domain-containing protein 1 1.64 RIN1 Ras and Rab interactor 1 1.63 CMAS N-acylneuraminate cytidylyltransfcrasc 1.63 NQO1 NAD(P)H dehydrogenase [quinone] 1 1.63 ERLEC1 Endoplasmic reticulum lectin 1 1.63 CDS2 Phosphatidate cytidylyltransferase 2 1.63 GLUD2 Glutamate dehydrogenase 2, mitochondrial 1.62 VDAC1 Voltage-dependent anion-selective channel protein 1 1.61 TTC19 Tetratricopeptide repeat protein 19, mitochondrial 1.61 SEMA3C Semaphorin-3C 1.61 LRSAM1 E3 ubiquitin-protein ligase LRSAM1 1.60 ACOT13 Acyl-coenzyme A thioesterase 13 1.60 LXN Latexin 1.60 GSN Gelsolin 1.60 CHP1 Calcineurin B homologous protein 1 1.60 GALNT2 N-acetylgalactosaminyltransferase 2 1.60 RARS2 Arginine-tRNA ligase, mitochondrial 1.60 PACS1 Phosphofurin acidic cluster sorting protein 1 1.60 RMDN3 Regulator of microtubule dynamics protein 3 1.60 PANK4 Pantothenate kinase 4 1.59 KTN1 Kinectin 1.59 CTSB Cathepsin B 1.58 BCKDHA 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial 1.58 EBAG9 Receptor-binding cancer antigen expressed on SiSo cells 1.58 TMEM214 Transmembrane protein 214 1.58 UQCC2 Ubiquinol-cytochrome-c reductase complex assembly factor 2, mitochondrial 1.58 TM9SF4 Transmembrane 9 superfamily member 4 1.58 HDHD2 Haloacid dehalogenase-like hydrolase domain-containing protein 2 1.58 EPHX1 Epoxide hydrolase 1 1.58 TMF1 TATA element modulatory factor 1.58 CDIPT CDP-diacylglyecrol-inositol 3-phosphatidyltransferase 1.57 CD81 CD81 antigen 1.57 SRXN1 Sulfiredoxin-1 1.57 ME1 NADP-dependent malic enzyme 1.57 ACOT8 Acyl-coenzyme A thioesterase 8, peroxisomal 1.57 SMDT1 Essential MCU regulator, mitochondrial 1.56 ALG1 Chitobiosyldiphosphodolichol beta-mannosyltransferase 1.56 DNAJC5 DnaJ homolog subfamily C member 5 1.55 DBT Lipoamide acyltransferase component of branched-chain alpha-keto acid 1.55 dehydrogenase complex, mitochondrial LAMTOR2 Ragulator complex protein LAMTOR2 1.54 TIGAR Fructose-2,6-bisphosphatase TIGAR 1.54 IDUA Alpha-L-iduronidase 1.54 TMEM87B Transmembrane protein 87B 1.54 TNKS1BP1 182 kDa tankyrase-1-binding protein 1.54 MIA3 Transport and Golgi organization protein 1 homolog 1.54 TXNRD1 Thioredoxin reductase 1, cytoplasmic 1.54 MYOF Myoferlin 1.54 RABEP2 Rab GTPase-binding effector protein 2 1.53 GLUD1 Glutamate dehydrogenase 1, mitochondrial 1.53 PDF Peptide deformylase, mitochondrial 1.53 TAPBP Tapasin 1.53 NDUFS7 NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial 1.53 ATP2C1 Calcium-transporting ATPase type 2C member 1 1.53 ANK3 Ankyrin-3 1.53 ABHD11 Protein ABHD11 1.53 AGO3 Protein argonaute-3 1.53 S100A16 Protein S100-A16 1.53 TM7SF2 Delta(14)-sterol reductase 1.53 MRPL21 39S ribosomal protein L21, mitochondrial 1.53 RAB9A Ras-related protein Rab-9A 1.53 TOM1 Target of Myb protein 1 1.53 C21orf33 ES1 protein homolog, mitochondrial 1.52 SURF1 Surfeit locus 1 (cytochrome c oxidase assembly protein), mitochondrial 1.52 NAMPT Nicotinamide phosphoribosyltransferase 1.51 METTL7B Methyltransferase-like protein 7B 1.51 CTSA Cathepsin A 1.51 TTC37 Tetratricopeptide repeat protein 37 1.51 RIDA 2-iminobutanoate/2-iminopropanoate deaminase 1.50 ARPC1A Actin-related protein 2/3 complex subunit 1A 1.50 OS9 Protein OS-9 1.50 FUCA1 Tissue alpha-L-fucosidase 1.50
indicates data missing or illegible when filed
TABLE-US-00005 TABLE 5 Mitochondrial-related Proteins Up-regulated in e-CSCs, derived from MCF7 3D-Spheroids Fold-Change Symbol Gene Description (Up-regulation) GLRX Glutaredoxin-1 10.79 ALDH3A1 Aldehyde dehydrogenase, dimeric NADP-preferring 10.24 QPRT Nicotinate-nucleotide pyrophosphorylase [carboxylating] 3.72 MIC13 MICOS complex subunit MIC13, mitochondrial 2.36 OXSM 3-oxoacyl-[acyl-carrier-protein] synthase, mitochondrial 2.22 FECH Ferrochelatase, mitochondrial 2.16 MCU Calcium uniporter protein, mitochondrial 2.13 GCLC Glutamate-cysteine ligase catalytic subunit 2.01 NQO2 Ribosyldihydronicotinamide dehydrogenase [quinone] 1.90 CMC1 COX assembly mitochondrial protein homolog 1.87 ISCU Iron-sulfur cluster assembly enzyme ISCU, mitochondrial 1.83 ABAT 4-aminobutyrate aminotransferase, mitochondrial 1.81 ABCB6 ATP-binding cassette sub-family B member 6, mitochondrial 1.80 ACADSB Short/branched chain specific acyl-CoA dehydrogenase, 1.79 mitochondrial COQ9 Ubiquinone biosynthesis protein COQ9, mitochondrial 1.77 ALDH5A1 Succinate-semialdehyde dehydrogenase, mitochondrial 1.76 CPT2 Carnitine O-palmitoyltransferase 2, mitochondrial 1.76 HMGCL Hydroxymethylglutaryl-CoA lyase, mitochondrial 1.75 FDXR NADPH:adrenodoxin oxidoreductase, mitochondrial 1.75 CYCS Cytochrome c 1.74 SQOR Sulfide:quinone oxidoreductase, mitochondrial 1.73 HMOX1 Heme oxygenase 1 1.69 MLYCD Malonyl-CoA decarboxylase, mitochondrial 1.67 ENDOG Endonuclease G, mitochondrial 1.67 IVD Isovaleryl-CoA dehydrogenase, mitochondrial 1.66 MRPS18C 28S ribosomal protein S18c, mitochondrial 1.66 HIBCH 3-hydroxyisobutyryl-CoA hydrolase, mitochondrial 1.66 MSRB2 Methionine-R-sulfoxide reductase B2, mitochondrial 1.64 NQO1 NAD(P)H dehydrogenase [quinone] 1 1.63 GLUD2 Glutamate dehydrogenase 2, mitochondrial 1.62 VDAC1 Voltage-dependent anion-selective channel protein 1 1.61 TTC19 Tetratricopeptide repeat protein 19, mitochondrial 1.61 ACOT13 Acyl-coenzyme A thioesterase 13, mitochondrial 1.60 RARS2 Arginine-tRNA ligase, mitochondrial 1.60 BCKDHA 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial 1.58 UQCC2 Ubiquinol-cytochrome-c reductase complex assembly factor 2, 1.58 Mitochondrial ME1 NADP-dependent malic enzyme 1.57 SMDT1 Essential MCU regulator, mitochondrial 1.56 DNAJC5 DnaJ homolog subfamily C member 5 1.55 DBT Lipoamide acyltransferase/branched-chain α-keto dehydrogenase, 1.55 mitochondrial TIGAR Fructose-2,6-bisphosphatase TIGAR 1.54 GLUD1 Glutamate dehydrogenase 1, mitochondrial 1.53 PDF Peptide deformylase, mitochondrial 1.53 NDUFS7 NADU dehydrogenase [ubiquinone] iron-sulfur protein 7, 1.53 Mitochondrial MRPL21 39S ribosomal protein L21, mitochondrial 1.53 C21orf33 ES1 protein homolog, mitochondrial 1.52 SURF1 Surfeit locus 1 (cytochrome c oxidase assembly protein), 1.52 Mitochondrial NAMPT Nicotinamide phosphoribosyltransferase 1.51
TABLE-US-00006 TABLE 6 Functional Markers of the e-CSC Phenotype (from MCF7 3D-Spheroids) Fold-Change Symbol Gene Description (Up-regulation) Senescence Markers CDKN1A Cyclin-dependent kinase inhibitor 1 (p21-WAF/CDK-inhihilor) 17.22 GLB1 Beta-galactosidase 1.64 Anti-Oxidant Response to ROS/Oxidative Stress GLRX Glutaredoxin-1 10.79 GCLC Glutamate-cysteine ligase catalytic subunit 2.01 NQO2 Ribosyldihydronicotinamide dehydrogenase [quinone] 1.90 MGST1 Microsomal glutathione S-transferase 1 1.88 MGST3 Microsomal glutathione S-transferase 3 1.84 SPATA20 Spermatogenesis-associated protein 20 (thioredoxin-like) 1.77 GSTK1 Glutathione S-transfcrasc kappa 1 1.75 NQO1 NAD(P)H dehydrogenase [quinone] 1 1.63 Stemness & Drug-Resistance/Radio-Resistance BCAS1 Breast carcinoma-amplified sequence 1 119.37 ALDH3A1 Aldehyde dehydrogenase, dimeric NADP-preferring 10.24 CEACAM6 Carcinoembryonic antigen-related cell adhesion molecule 6 9.66 CEACAM5 Carcinoembryonic antigen-related cell adhesion molecule 5 3.38 LAMA5 Laminin subunit alpha-5 2 72 ALDH5A1 Succinate-semialdehyde dehydrogenase, mitochondrial 1.76 CD44 CD44 antigen 1.65 Cytoskeletal Proteins (indicative of an EMT in CSCs) TUBA4A Tubulin alpha-4A chain 2.60 STOM Erythrocyte band 7 integral membrane protein 2.18 MAPRE3 Microtubule-associated protein RP/EB family member 3 1.74 KIF1A Kinesin-like protein KIF1A 1.66 RMDN3 Regulator of microtubule dynamics protein 3 1.60 GSN Gelsolin 1.60 MYOF Myoferlin 1.54 ANK3 Ankyrin-3 1.53 ARPC1A Actin-related protein 2/3 complex subunit 1A 1.50 Spindle Orientation and Mitotic Progression MISP Mitotic interactor and substrate of PLK1 1.66 Mitochondrial Biogenesis GLRX Glutaredoxin-1 10.79 MIC13 MICOS complex subunit MIC13, mitochondrial 2.36 OXSM 3-oxoacyl-[acyl-carrier-protein] synthase, mitochondrial 2.22 FECH Ferrochelatase, mitochondrial 2.16 CMC1 COX assembly mitochondrial protein homolog 1.87 ISCU Iron-sulfur cluster assembly enzyme ISCU, mitochondrial 1.83 COQ9 Ubiquinone biosynthesis protein COQ9, mitochondrial 1.77 HMOX1 Heme oxygenase 1 1.69 UQCC2 Ubiquinol-cytochrome-c reductase complex assembly factor 2, 1.58 Mitochondrial MRPS18C 28S ribosomal protein S18c, mitochondrial 1.66 RARS2 Arginine-tRNA ligase, mitochondrial 1.60 MRPL21 39S ribosomal protein L21, mitochondrial 1.53 PDF Peptide deformylase, mitochondrial 1.53 Glutamine/Asparagine Metabolism AGA N(4)-(beta-N-acctylglucosaminyl)-L-asparaginasc 2.30 GLUD2 Glutamate dehydrogenase 2, mitochondrial 1.62 GLUD1 Glutamate dehydrogenase 1, mitochondrial 1.53 NADH/NADPH: Synthesis & Salvage Pathway ALDH3A1 Aldehyde dehydrogenase, dimeric NADP-preferring 10.24 QPRT Nicotinate-nucleotide pyrophosphorylase [carboxylating] 3.72 RRM2 Ribonucleoside-diphosphate reductase subunit M2 2.34 ALDH5A1 Succinate-semialdehyde dehydrogenase, mitochondrial 1.76 FDXR NADPH:adrenodoxin oxidoreductase, mitochondrial 1.75 RRM2B Ribonucleoside-diphosphate reductase subunit M2 B 1.68 ME1 NADP-dependent malic enzyme 1.57 TIGAR Fructosc-2,6-bisphosphatasc TIGAR 1.54 TNKS1BP1 182 kDa tankyrase-1-binding protein 1.54 NDUFS7 NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, 1.53 mitochondrial NAMPT Nicotinamide phosphoribosyltransferase 1.51 Flavin-containing Enzymes CYP1A1 Cytochrome P450 1A1 6.60 MAOA Amine oxidase [flavin-containing] A 4.73 MAOB Amine oxidase [flavin-containing) B 2.45 GFER FAD-linked sulfhydryl oxidase ALR 1.80 CYB5R1 NADH-cytochrome b5 reductase 1 1.66 TXNRD1 Thioredoxin reductase 1, cytoplasmic (Glutaredoxin activity; 1.54 flavin-dependent) Epithelial Markers KRT10 Keratin, type 1 cytoskeletal 10 4.59 DPP7 Dipeplidyl peptidase 2 2.43 MUC5B Mucin-5B 1.98 Cell Surface Markers GM2A Ganglioside GM2 activator 2.36 CD59 CD59 glycoprotein 1.80 ENTPD8 Ectonucleoside triphosphate diphosphohydrolase 8 1.66 CD81 CD81 antigen 1.57 S100 Proteins S100P Protein S100-P 2.11 S100A10 Protein S100-A10 1.96 S100A16 Protein S100-A16 1.53 Autophagy/Lysosomes CHMP6 Charged multivesicular body protein 6 2.62 SERPINA3 Alpha-1-antichymotrypsin 2.33 CTSH Cathepsin H 1.98 GNS N-acetylglucosamine-6-sulfatase 1.97 GAA Lysosomal alpha-glucosidase 1.83 GALNS N-acetylgalactosamine-6-sulfatase 1.82 ATP6V0A1 V-type proton ATPase 116 kDa subunit a isoform 1 1.71 CTSD Cathepsin D 1.66 CPD Carboxypeptidase D 1.64 GALNT2 N-acetylgalactosaminyltransferase 2 1.60 CTSB Cathepsin B 1.58 CTSA Cathepsin A 1.51 Peroxisomes PEX14 Peroxisomal membrane protein PEX14 1.91 DECR2 Peroxisomal 2,4-dienoyl-CoA reductase 1.81 PEX11B Peroxisomal membrane protein PEX11B 1.76 ACOT8 Acyl-coenzyme A thioesterase 8, peroxisomal 1.57 RABs RAB27B Ras-related protein Rab-27B 2.69 RAB27A Ras-related protein Rab-27A 2.54 RAB24 Ras-related protein Rab-24 1.87 RIN1 Ras and Rab interactor 1 1.63 RABEP2 Rab GTPase-binding effector protein 2 1.53 RAB9A Ras-related protein Rab-9A 1.53 Annexins and PARP ANXA1 Annexin A1 2.87 PARP4 Poly [ADP-ribose] polymerase 4 2.55 ANXA2 Annexin A2 1.65 Calcium/Calmodulin CIB1 Calcium and integrin-binding protein 1 2.90 MCE Calcium uniporter protein, mitochondrial 2.13 CAPS Calcyphosin 1.79 CAMK2D Calcium/calmodulin-dependent protein kinase, type II 1.70 subunit delta CALML5 Calmodulin-like protein 5 1.69 TACSTD2 Tumor-associated calcium signal transducer 2 1.64 CHP1 Calcineurin B homologous protein 1 1.60 SMDT1 Essential MCU regulator, mitochondrial 1.56 ATP2C1 Calcium-transporting ATPase type 2C member 1 1.53
TABLE-US-00007 TABLE 7 eCSC Marker Proteins are Transcriptionally Up-regulated in Patient- derived Human Breast Cancer Cells in Vivo Fold- Symbol Gene Description Change P-value TSPAN31 Tetraspanin-31 4.72 8.45E−06 CDS2 Phosphatidate cytidylyltransferase 2 4.71 8.73E−06 PEX11B Peroxisomal membrane protein PEX11B 4.69 9.58E−06 RAB9A Ras-related protein Rab-9A 4.47 2.02E−05 TACSTD2 Tumor-associated calcium signal 4.41 2.47E−05 transducer 2 GLUD1 Glutamate dehydrogenase 1, 4.38 2.76E−05 mitochondrial MSRB2 Methionine-R-sulfoxide reductase 4.31 3.49E−05 B2, mitochondrial SURF1 Surfeit locus 1 (cytochrome c oxidase 4.16 5.66E−05 assembly protein), mitochondrial PON2 Serum paraoxonase/arylesterase 2 4.01 9.25E−05 CYB5R1 NADH-cytochrome b5 reductase 1 3.94 1.18E−04 ANK3 Ankyrin-3 3.81 1.77E−04 ASAH1 Acid ceramidase 3.80 1.83E−04 CD59 CD59 glycoprotein 3.60 3.47E−04 OXSM 3-oxoacyl-[acyl-carrier-protein] 3.49 4.82E−04 synthase, mitochondrial NQO1 NAD(P)H dehydrogenase [quinone] 1 3.49 4.81E−04 SEMA3C Semaphorin-3C 3.49 4.92E−04 CD44 CD44 antigen 3.44 5.69E−04 ALDH5A1 Succinate-semialdehyde dehydrogenase, 3.43 5.75E−04 mitochondrial AGA N(4)-(beta-N-acetylglucosaminyl)-L- 3.40 6.30E−04 asparaginase GSTK1 Glutathione S-transferase kappa 1 3.39 6.59E−04 KTN1 Kinectin 3.36 7.16E−04 FECH Ferrochelatase, mitochondrial 3.36 7.20E−04 C21orf33 ES1 protein homolog, mitochondrial 3.31 8.40E−04 MPV17 Protein Mpv17 3.27 9.44E−04 TMEM214 Transmembrane protein 214 3.12 1.44E−03 NEBL Nebulette 3.09 1.59E−03 CDIPT CDP-diacylglycerol-inositol 3- 3.06 1.74E−03 phosphatidyltransferase CPT2 Carnitine O-palmitoyltransferase 2, 3.02 1.94E−03 mitochondrial ATP2C1 Calcium-transporting ATPase type 2C 3.01 1.96E−03 member 1 SERPINA3 Alpha-1-antichymotrypsin 2.99 2.11E−03 CYCS Cytochrome c 2.92 2.52E−03 TTC19 Tetratricopeptide repeat protein 19, 2.85 3.06E−03 mitochondrial SELENBP1 Methanethiol oxidase 2.84 3.22E−03 MIA3 Transport and Golgi organization 2.76 3.98E−03 protein 1 homolog OS9 Protein OS-9; amplified in osteosarcoma 2.76 3.99E−03 ANXA2 Annexin A2 2.73 4.30E−03 SULT1A1 Sulfotransferase 1A1 2.72 4.34E−03 MYOF Myoferlin 2.67 5.00E−03 CAPN2 Calpain-2 catalytic subunit 2.64 5.42E−03 VDAC1 Voltage-dependent anion-selective 2.64 5.35E−03 channel protein 1, mitochondrial TXNRD1 Thioredoxin reductase 1, cytoplasmic 2.64 5.36E−03 EPS8L1 Epidermal growth factor receptor kinase 2.57 6.54E−03 substrate 8-like protein 1 PDF Peptide deformylase, mitochondrial 2.56 6.71E−03 CTSH Cathepsin H 2.54 7.07E−03 KRT10 Keratin, type cytoskeletal 10 2.53 7.19E−03 GLB1 Beta-galactosidase 2.53 7.20E−03 GM2A Ganglioside GM2 activator 2.42 9.42E−03 RRM2 Ribonucleoside-diphosphate reductase 2.40 9.93E−03 subunit M2 RETSAT All-trans-retinol 13,14-reductase 2.39 1.03E−02 RNASET2 Ribonuclease T2 2.36 1.10E−02 ENDOG Endonuclease G, mitochondrial 2.32 1.22E−02 NAMPT Nicotinamide phosphoribosyltransferase 2.19 1.66E−02 SPATA20 Spermatogenesis-associated protein 20 2.16 1.77E−02 SLC22A18 Solute carrier family 22 member 18 2.14 1.86E−02 ABAT 4-aminobutyrate aminotransferase, 2.08 2.14E−02 mitochondrial TAPBP Tapasin 2.08 2.13E−02 CIB1 Calcium and integrin-binding protein 1 2.04 2.34E−02 HMGCL Hydroxymethylglutaryl-CoA lyase, 2.03 2.38E−02 mitochondrial FAMSA1 Protein FAM8A1 2.02 2.40E−02 GCLC Glutamate-cysteine ligase catalytic 2.01 2.49E−02 subunit ACAA1 3-ketoacyl-CoA thiolase, peroxisomal 2.00 2.53E−02 GLRX Glutaredoxin-1 1.92 3.01E−02 ISCU Iron-sulfur cluster assembly enzyme 1.92 3.02E−02 ISCU, mitochondrial TMF1 TATA element modulatory factor 1.88 3.25E−02 CD81 CD81 antigen 1.87 3.34E−02 NQO2 Ribosyldihydronicotinamide 1.79 3.98E−02 dehydrogenase [quinone] MAOB Amine oxidase [flavin-containing] B 1.74 4.41E−02 CEACAM6 Carcinoembryonic antigen-related cell 1.70 4.71E−02 adhesion molecule 6 SLC9A1 Sodium/hydrogen exchanger 1 1.68 4.97E−02
indicates data missing or illegible when filed
TABLE-US-00008 TABLE 8 Tumor Recurrence (RFS): Predicting Tamoxifen-resistance in ER(+) Breast Cancer Patients Gene Probe Gene Symbol HR (Hazard-Ratio) Log-Rank Test 201468_s_at NQO1 2.47 0.0023 203608_at ALDH5A1 2.21 0.01 201266_at TXNR 2.17 0.0062 201890_at RRM2 2.54 0.00089 Combined Signature (RFS) 3.89 4.1e−05 RFS, recurrence-free survival.
TABLE-US-00009 TABLE 9 Distant Metastasis (DMFS): Predicting Tamoxifen- resistance in ER(+) Breast Cancer Patients Gene Probe Gene Symbol HR (Hazard-Ratio) Log-Rank Test 201468_s_at NQO1 1.73 0.1 203608_at ALDH5A1 2.86 0.0034 201266_at TXNR 3.64 0.00035 201890_at RRM2 3.02 0.00092 DMFS, distant metastasis-free survival.