LON PROTEASE, ALPHA-HEMOLYSIN, CK1-ALPHA-1; C-MYB INHIBITOR OR A CEBP-DELTA INHIBITOR AS THERAPEUTICS
20230055209 · 2023-02-23
Assignee
Inventors
Cpc classification
A61P31/00
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61P13/02
HUMAN NECESSITIES
C12N9/12
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61P13/02
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
A therapeutic agent comprising Lon protease, or a variant or active fragment thereof, alpha-hemolysin, or a variant or active fragment thereof, CK1α1, or a variant or active fragment thereof, a c-MYB inhibitor and/or a CEBP-δ inhibitor, for use in therapy, with the proviso that the therapeutic agent does not comprise a bacteria or bacterial supernatant. Methods of production and use thereof.
Claims
1. A therapeutic agent comprising Lon protease, or a variant or active fragment or equivalent thereof; alpha-hemolysin, or a variant or active fragment or equivalent thereof; CK1α1, or a variant or active fragment or equivalent thereof; a c-MYB inhibitor or a CEBP-δ inhibitor; or combination thereof, for use in therapy, with the proviso that the therapeutic agent does not comprise a bacteria or bacterial supernatant or lysate.
2. A therapeutic agent for use according to claim 1, wherein the Lon protease, alpha-hemolysin, c-MYB inhibitor and/or CEBP-δ inhibitor are bacterial, preferably wherein the bacteria is E. coli, more preferably E. coli 536 or CFT073.
3. A therapeutic agent for use according to any preceding claim, comprising isolated Lon protease, or a variant or active fragment or equivalent thereof, isolated alpha-hemolysin, or a variant or active fragment or equivalent thereof, isolated CK1α1, or a variant or active fragment or equivalent thereof, an isolated c-MYB inhibitor and/or an isolated CEBP-δ inhibitor.
4. A therapeutic agent for use according to any preceding claim, comprising synthetic Lon protease, or a variant or active fragment or equivalent thereof, synthetic alpha-hemolysin, or a variant or active fragment or equivalent thereof, synthetic CK1α1, or a variant or active fragment or equivalent thereof, a synthetic c-MYB inhibitor and/or a synthetic CEBP-δ inhibitor
5. A therapeutic agent for use according to any preceding claim, wherein the c-MYB inhibitor and/or CEBP-δ inhibitor are proteins or variants or active fragments thereof.
6. A therapeutic agent for use according to any preceding claim, wherein the use is treatment or prevention of a disease wherein c-MYC levels are elevated.
7. A therapeutic agent for use according to any preceding claim, wherein the use is treatment or prevention of cancer and/or infection, preferably wherein the cancer is lymphoma, epithelial cancer or mucosal cancer, preferably wherein the infection is of the urinary tract.
8. A therapeutic agent for use according to any preceding claim, wherein the c-MYB inhibitor and/or a CEBP-δ inhibitor are inhibitors of c-MYB and/or CEBP-δ expression.
9. A recombinant microorganism engineered to express elevated levels of Lon protease, or a variant or active fragment or equivalent thereof, alpha-hemolysin, or a variant or active fragment or equivalent thereof, CK1α1, or a variant or active fragment or equivalent thereof, a c-MYB inhibitor and/or a CEBP-δ inhibitor.
10. A microorganism extract obtained from a recombinant microorganism according to claim 10.
11. A method of obtaining isolated Lon protease, or a variant or active fragment or equivalent thereof, isolated alpha-hemolysin, or a variant or active fragment or equivalent thereof, isolated CK1α1, or a variant or active fragment or equivalent thereof, an isolated c-MYB inhibitor and/or an isolated CEBP-δ inhibitor, the method comprising isolating the Lon protease, or a variant or active fragment or equivalent thereof, the alpha-hemolysin, or a variant or active fragment or equivalent thereof, the CK1α1, or a variant or active fragment or equivalent thereof, the c-MYB inhibitor and/or the CEBP-δ inhibitor from the recombinant microorganism of claim 9 or the microorganism extract of claim 10.
12. A kit comprising isolated Lon protease, or a variant or active fragment or equivalent thereof, isolated alpha-hemolysin, or a variant or active fragment or equivalent thereof, isolated CK1α1, or a variant or active fragment or equivalent thereof, an isolated c-MYB inhibitor and/or an isolated CEBP-δ inhibitor.
13. A kit according to claim 12, comprising synthetic Lon protease, or a variant or active fragment or equivalent thereof, synthetic alpha-hemolysin, or a variant or active fragment or equivalent thereof, synthetic CK1α1, or a variant or active fragment or equivalent thereof, a synthetic c-MYB inhibitor and/or a synthetic CEBP-δ inhibitor.
14. A kit according to claim 12 or 13, wherein the Lon protease, or variant or active fragment or equivalent thereof, alpha-hemolysin, or variant or active fragment or equivalent thereof, CK1α1, or a variant or active fragment or equivalent thereof, c-MYB inhibitor and/or CEBP-δ inhibitor are in powder form.
15. A recombinant microorganism according to claim 9, a recombinant microorganism extract according to claim 10, or a kit according to any of claims 12-14, for use in therapy.
16. A pharmaceutical composition, comprising a therapeutic agent according to any of claims 1-8, a recombinant microorganism according to claim 9, or a microorganism extract according to claim 10, plus a pharmaceutically acceptable carrier, excipient and/or adjuvant.
17. A method of treating cancer and/or infection, the method comprising administering an effective amount of a therapeutic agent according to any of claims 1-8, a recombinant microorganism according to claim 9, a microorganism extract according to claim 10, or a pharmaceutical composition according to claim 16, to a subject in need thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:
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[0068] c-MYC protein levels in A498 cells infected with uropathogenic strains E. coli CFT073 or E. coli 536 or asymptomatic bacteriuria (ABU) strain E. coli 83972 (10.sup.5 CFU/ml, 1 or 4 hours), relative to PBS control. a, Confocal images of infected cells (Scale bars=10 μm). b, Quantification of c-MYC levels based on fluorescence intensity from (a), n=3 experiments. c, Representative Western blot (WB) showing a reduction in c-MYC levels in extracts from infected cells, n=3 experiments. d, c-MYC levels in cells infected with acute pyelonephritis (APN) (n=36) or ABU (n=20) isolates, means of 2 experiments per strain. Quantification of c-MYC levels by WB of infected cell extracts, 4 hours). Histogram inset shows group means. For cell viability data, see
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[0070] a, Schematic representation of the E. coli 536 chromosome, indicating the positions of pathogenicity islands (PAIs) I to V. b-c, E. coli 536 deletion mutants (PAI I.sub.536 to PAI V.sub.536 and PAI I-II.sub.536) were screened for effects on cellular c-MYC levels in A498 cells (10.sup.5 cfu/ml, 4 hours) using Confocal microscopy (b) and WB analysis (c) of infected cell extracts. PAI I.sub.536 was identified as a loss-of-function mutant (Scale bars=10 μm. See also
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[0072] a, Table of proteins detected in the supernatant of E. coli 536 but not in PAI I.sub.536. Hly and the Lon protease were selected for further study (LC-MS/MS, see also
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[0074] a, Gene expression analysis identifies of A498 cells infected with E. coli 536 or PAI I.sub.536. Inversely regulated genes are identified. Red: upregulated, blue: down-regulated, black: not significantly regulated (cut-off fold change ≥1.41). b, A subset of MYB-related genes was inversely regulated. c, Confocal imaging of c-MYB protein levels in infected cells and quantification compared to cells exposed to the bacterial supernatant. Scale bar, 10 μm. d, MYB mRNA levels in cells exposed to E. coli 536 bacteria or supernatant, compared to PAI I.sub.536. (n=4). e, Confocal imaging of CEBPD protein levels in infected cells and quantification compared to cells exposed to the bacterial supernatant. Scale bar, 10 μm. f, CEBPD mRNA levels in cells exposed to E. coli 536 bacteria or supernatant, compared to PAI I.sub.536. (n=4). g, MYC and MAX expression levels in kidney epithelial cells infected with E. coli 536 or PAI I.sub.536 (qRT-PCR, n=4 experiments). h, MAX protein staining in infected cells (confocal microscopy). i, Quantification of fluorescence intensity in h, (n=2). j, MYB and CEBPD expression in patients with acute pyelonephritis, compared to paired follow-up samples. MYB levels were reduced during acute infection. k, Model of c-MYC regulation by bacteria. Three mechanisms of c-MYC degradation have been uncovered. (i) Degradation by the bacterial Lon protease. (ii) Activation of CK1α1, by α-hemolysin-dependent Ca.sup.2+ fluxes, targeting c-MYC for proteasomal degradation. (iii) c-MYB degradation by CK1α1 activation and transcriptional repression of CEPBD, suggesting mechanisms, which may lead to inhibited c-MYC expression. Data is presented as means±SEMs and analysed by paired t-test (c-i) or Wilcoxon matched pairs signed rank test (j). *P<0.05, **P<0.01.
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[0076] a, c-MYC inhibition by E. coli 536 infection in Burkitt lymphoma cells (Daudi and Raji) and human or mouse bladder- (HTB-9 and MB49), lung- (A549), gastric- (AGS), colon- (DLD1) and hepatic (HEP-G2) carcinoma cells. b, MYC inhibition by E. coli 536 supernatants in HTB9, A549, DLD1 and MB49 cells but not by PAI I.sub.536, 536Δhly.sup.1/2 or 536Δlon supernatants (See also
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[0079] A498 cells were infected with E. coli 83972 (ABU), E. coli CFT073 (APN) or E. coli 536 (APN), (10.sup.5 CFU/ml). PBS served as a control. Cell viability was measured by (a) Lactate dehydrogenase (LDH) release and (b) Presto Blue levels.
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[0081] A498 cells were infected with different E. coli 536 deletion mutants (PAI I.sub.536 to PAI V.sub.536 and PAI I-II.sub.536) and screened for effects on cellular c-MYC levels (10.sup.5 CFU/ml, 4 hours). a, Low magnification confocal images. b, Quantification of fluorescent intensity from (a). Data is presented as mean±SEM from n=3 experiments and analyzed by paired t-test. **P<0.01, ***P<0.001.
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[0083] A single colony from each E. coli strain or mutant was cultured in Luria Broth in a shaking incubator at 37° C. OD.sub.600 was measured at 1, 2, 3, 4, 6 and 8h. All growth curves were replicates, two experiments per strain.
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[0085] a, Comparative Mass spec analysis of supernatants from E. coli 536 and PAI I.sub.536. Five bands, abundant in E. coli 536 supernatant (SN) (bands 1-5, Coomassie staining), were excised, processed by in-gel digestion and analyzed by LC-MS/MS. b, The 10 most abundant proteins from each excised band are listed. c, A498 kidney epithelial cells were pre-treated with inhibitors of ATPase or GTPase driven endocytosis (Quercetin [25 μM] and Dynasore [30 μM] respectively) for 30 minutes prior to recombinant Lon protease (rLon) treatment (8 μg/ml, 4 hours). Confocal images show reduced Lon protease staining and uptake. d, Quantification of Lon protease staining in 50 cells (n=3 experiments). Data is presented as mean±SEM and analyzed by two-tailed t-test. *P<0.05. e, Protease digestion of E. coli 536 supernatant removed the MYC inhibitor, confirming that the soluble bacterial MYC inhibitor is a protein. Supernatants were pre-treated with increasing concentrations of trypsin (0-5 μg/ml, 30 minutes) before being added to A498 cells (4 hours, Western blot analysis). Trypsin was used as a negative control. f, Binding of recombinant Lon protease to recombinant c-MYC in a dose-dependent manner.
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[0087] In the control sample 23 specific peptides corresponding to 51% of the sequence were detected. In rLon degraded c-MYC, 10 specific peptides corresponding to 23% of the sequence were found.
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[0089] a, Genes involved in proteasome degradation were upregulated in E. coli 536 infected A498 kidney epithelial cells, including CSNK1A1 and PLK3. b, CSNK1A1 expression was increased by E. coli 536 infection as well as E. coli 536 culture supernatants (SNs) (n=4 experiments). c, CK1α1 activation, defined by a redistribution from the nuclei to characteristic cytoplasmic puncta. Scale bar, 10 μm. d, Binding of CK1α1 to c-MYC shown by co-immunoprecipitation, using recombinant c-MYC and lysates from infected cells (n=3 experiments). e-f, Reversal of c-MYC inhibition by the CK1α1 inhibitor IC261 (30 minutes pre-treatment) by e, confocal imaging (Scale bars=10 μm) and quantification (black=control, red=E. coli 536) f, Western blot analysis (n=4 experiments). g, To inhibit Ca.sup.2+ fluxes, cells were pre-treated with EGTA (50 with μg/ml, 30 minutes) prior to stimulation with supernatants from 536 WT or 536Dhly.sup.1/2. EGTA pre-treatment inhibited the activation of CK1α1. Data is represented as mean±SEM and analysed by paired t-tests. *P<0.05, **P<0.01, ***P<0.001.
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[0091] a, Quantification of c-MYC levels in cells infected with E. coli 536 or E. coli 536Dhly.sup.1/2 (top panels) or exposure to bacterial supernatants (bottom panels). b, Quantification of c-MYC fluorescence in A498 kidney epithelial cells (n=2 experiments, compared to uninfected cells). Green=c-MYC; blue=nuclei. Scale bar, 10 μm. c, Western blot analysis of the corresponding cell extracts. d-f, Removal of molecules >100 kDa from E. coli 536 supernatant by molecular size filtration. The molecular size of the α-hemolysin (HlyA) protein is 109 kDa, predicting that HlyA should be excluded from the <100 kDa fraction, which still inhibited c-MYC in human cells, as shown by confocal imaging in (d-e) and Western blots in (f-g), Fractions <100 kDa did not affect cell viability quantified by ATP lite (N.S., compared to untreated cells, n=3 experiments). Data is presented as mean±SEM and analyzed by student's t-test. *P<0.05, **P<0.01, ***P<0.001.
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[0093] The clinical ABU and APN isolates from the collection shown in
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[0095] A498 kidney epithelial cells were infected with E. coli 536, 536 Dhly.sup.1/2 or 536 Dlon (10.sup.5 CFU/ml, 4 hours) a, d, g, MAX levels were not affected. b, e, h, c-MYB levels were reduced by E. coli 536, and Dlon but not affecting mRNA levels. c, f, i, CEBPD levels were only affected by E. coli 536. Confocal images (target proteins=green, nuclei=blue). d-f Quantification of images in (a-c). d, MAX, e, c-MYB and f, C/EBP-δ staining intensity compared to PBS. g-i, (g) MAX, (h) c-MYB and (i) CEBPD mRNA levels quantified by qRT-PCR, PBS served as control. Data is presented as mean±SEM and was analyzed by paired t-test (n=2-3 experiments). *P<0.05, **P<0.01, ***P<0.001.
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[0097] a, A498 cells were infected with E. coli 536, PAI I.sub.536, E. coli 536 Dhly.sup.1/2 or E. coli 536 Dlon (10.sup.5 CFU/ml, 4 hours), or treated with supernatants. WB of c-MYC levels. b, Cell viability as determined by LDH release after in vitro infection of A498 kidney epithelial cells. c, No change in viability in cells exposed to increasing concentrations of recombinant Lon protease (rLon) determined by LDH, ATP lite or Presto Blue.
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[0099] a, Tumor development was compared on day 12 between mice receiving E. coli 536 or PAI I.sub.536 supernatants (SN). Bladder size and b, Tumor size, quantified from whole bladder tissue mounts, representative images from two mice. c, c-MYC expression was reduced in mice treated with E. coli 536 supernatant compared to mice receiving PBS or PAI I.sub.536 supernatant. d, Western blot analysis of whole bladder tissue extracts in (c) (n=5 per group). e, Transcriptomic analysis of whole bladder RNA. The shift from a disease-specific towards a healthy tissue profile in E. coli 536 treated mice was not seen in the PAI I.sub.536 group. f, Quantification of bladder weight, bladder size, tumor size and MYC staining. Each dot indicates a separate mouse and the lines represent medians. Data from sham and E. coli 536 supernatant treated mice is also included in main
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[0101] a, Heat map of regulated genes in tumor bearing mice on day 12 treated with E. coli 536 supernatant (SN)-, E. coli PAI I.sub.536 supernatant- or sham treated controls, compared to healthy bladders. A marked reduction in the number of regulated genes was observed in the E. coli 536 supernatant group (Heat map, FC >2.0, n=1 mouse per group). b, Activation of MYC-dependent network by E. coli PAI I.sub.536 supernatant treated and Sham mice. c, Further analyses showed strong inhibition of cancer pathways in the E. coli 536 supernatant treated mice, compared to the sham or E. coli PAI I.sub.536 supernatant groups.
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[0103] a, Significantly regulated gene categories, showing strong regulation of Cancer related categories. b, Heat map of regulated genes in tumor bearing mice treated with E. coli 536 supernatant (SN)- or recombinant Lon protease (rLon)-treated mice and the sham group, compared to healthy bladders. c, Principle Component Analysis (PCA) plot showing a shift from disease to a healthier phenotype in rLon and E. coli 536 supernatant-treated mice. d, Cancer functions regulated in the E. coli 536 supernatant treated mice, compared to the sham group. e, MYC-related network in E. coli 536 supernatant treated mice compared to the sham group. f, Biofunctions regulated by E. coli 536 supernatant or rLon compared to healthy bladders. g, Corresponding Z-scores for significantly regulated functions. Data from rLon compared to Sham is also included in
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[0105] Therapeutic effect of E. coli 536 supernatant (SN) treatment in APC.sup.Min/+ mice compared to the sham group, showing a reduction in intestinal polyps in a, 13 weeks old mice, b, 22 weeks old mice. Each dot represents individual mice and the line show the median and was analyzed by Mann-Whitney U-test. c, MYC-dependent gene network analysis in 13 weeks old APC.sup.Min/+ mice compared to healthy C57BL/6 WT mice, confirming the c-MYC dependence of intestinal tumorigenesis in APC.sup.Min/+ mice. d, Inhibition of MYC-dependent gene networks in mice treated with E. coli 536 supernatant, compared to sham treated mice. Data from rLon compared to Sham is also included in
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[0107] a, Kaplan Meyer plot showing increased survival in recombinant Lon protease (rLon) treated mice. b, Body weight in sham mice (12 days), rLon treated mice without further intervention (27 days, follow-up) and rLon treated mice with twice weekly treatment until sacrifice (35 days, long-term). c-e, Changes in bladder pathology, bladder weight and bladder size in rLon treated mice. f, Heatmap of regulated genes in the sham group, rLon treated mice follow-up and long-term groups compared to healthy mice (n=2). g, Genes associated with the Molecular Mechanisms of Cancer pathways were strongly inhibited by rLon treatment. The activation was increasingly lower in follow-up and long-term treated mice, respectively.
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[0109] a, Pie chart of significantly regulated gene categories by recombinant Lon protease (rLon) treatment. b, Details of cancer-related functions regulated in rLon treated mice compared to the sham group (W25). c, Network showing inhibition of MYC-related genes. The MYC inhibition was maintained in the long-term follow-up group.
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[0111] a, C57BL/6J WT mice were exposed to recombinant Lon protease (rLon) intravesically for 12 days. No change in bladder weight or macroscopic appearance of the bladder was detected. b, Few genes were regulated in bladders from healthy rLon treated mice (n=2 rLon, 1 healthy). c, MYC expression was not regulated. A small number of MYC-dependent genes was regulated in bladders from healthy rLon treated mice (33 activated, 1 inhibited).
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[0113] a, C57BL/6J WT mice were exposed to recombinant Lon protease (rLon) per-orally twice daily for 14 days. No change in body weight, or macroscopic appearance of the intestine, liver, kidney or spleen was detected. b, Gene expression analysis detected a lack of transcriptional activation in healthy rLon treated mice (n=3 rLon, 1 healthy). c, MYC expression was not regulated and few MYC-related genes were affected in healthy intestines after rLon challenge (1 activated and 8 inhibited genes).
DETAILED DESCRIPTION
[0114] In experimental cancer models, c-Myc inhibition has already been shown to arrest cancer progression and even restore tissue integrity. Here, we identify a new molecular strategy for MYC inhibition, developed by pathogenic E. coli strains. Based on a combination of accelerated degradation and inhibition of gene expression, the bacteria effectively reduce MYC levels in infected cells and tissues.
Example 1—Rapid Depletion of Renal c-MYC Levels Following Infection
[0115] To investigate if renal c-MYC expression is modified by infection, we selected human kidney epithelial cells, which are the first to be targeted when bacteria ascend into the renal pelvis. A rapid, time-dependent reduction in c-MYC protein levels was detected after infection with E. coli CFT073; a uropathogenic strain (
[0116] c-MYC staining was also reduced in vivo, after intra-vesical infection of mice with CFT073 (P<0.05,
Example 2—Gene Deletion Strategy to Identify Bacterial c-MYC Regulators/Inhibitors
[0117] To address the mechanism of bacterial c-MYC inhibition, we selected E. coli 536, in which virulence genes and chromosomal pathogenicity islands (PAIs) have been extensively characterized (
[0118] By sequential deletions from the 5′ end of PAI I using lambda red homologous recombination, we identified a bacterial ABC metal ion transporter encoded on the PAI I 2.5.3 sub-fragment (ORF42-ORF46). The ABC transporter controlled MYC inhibition in E. coli 536 and by deletion of the corresponding ABC transporter in CFT073, which in this strain is located outside of PAI I, we confirmed its importance for bacterial MYC inhibition (
[0119] The inhibitory effects of E. coli 536 and CFT073 were reproduced, using bacterial culture supernatants (RPMI, 4 hours), suggesting that bacteria release molecules to achieve this effect. ZnCl.sub.2 abolished this effect indicating that Zn.sup.2+ regulates the production of the bacterial MYC inhibitors. In contrast, ZnCl.sub.2 did not affect the execution of MYC suppression in host cells (
Example 3—Mechanism of MYC Degradation in Host Cells
[0120] Transcription factors are rapidly degraded and c-MYC has an estimated half-life of around 30 minutes. We have obtained convincing evidence that bacteria accelerate MYC protein degradation and inhibit MYC expression.
[0121] Two separate mechanisms of MYC degradation were defined. By comparative mass spectrometry of supernatants from E. coli 536 and ΔPAI I loss of function mutant, we identified the Lon serine protease as an abundant constituent of E. coli 536 supernatant (
[0122] By gene expression analysis of infected cells, we also observed a set of proteasome related genes, which were inversely regulated, by E. coli 536 or ΔPAI I (
[0123] The results suggest that E. coli 536 accelerates c-MYC degradation by at least two distinct mechanisms, defined by uptake of the bacterial Lon protease and activation of the human CK1α1 kinase.
Example 4—Bacteria Inhibit c-MYC Expression
[0124] MYC expression was reduced in infected human kidney cells, suggesting that the bacteria also affect c-MYC expression and the supernatants reproduced these effects (4 hours,
[0125] Transcriptomic analysis defined a network of MYB (myeloblastosis)-related genes, which was inhibited in E. coli 536 infected cells (
[0126] Biological Relevance
[0127] The MYC family of oncogenes is subject to “a bewildering assortment of genetic rearrangements” associated with many different tumors cell types. We therefore investigated, if the bacterial MYC inhibition mechanism is relevant also for cancer cells of other origins. E. coli 536 infection reduced cellular c-MYC levels in Burkitt lymphoma cells, where c-MYC was first discovered, as well as gastric (AGS) and hepatic (HEP-G2) carcinoma cells (
[0128] Therapeutic effects of the bacterial supernatant were demonstrated in the murine MB49 bladder cancer model. Bladder cancer was established in C57BL/6 mice, by instillation of MB49 cells and the treatment group received five intra-vesical instillations of E. coli 536 supernatant, ΔPAI I supernatant or PBS (sham) (
[0129] Materials and Methods for Examples 1 to 4
[0130] This study addressed the mechanism of c-MYC control by uropathogenic E. coli. The study was initiated when a transcriptomic screen of patients prone to acute pyelonephritis (APN) revealed elevated expression of MYC and MYC-related genes compared to patients with asymptomatic bacteriuria (ABU). As RNA was collected during an infection free interval, these differences were not likely to reflect a direct response to infection but a difference in baseline MYC activity. To understand the role of c-MYC during acute pyelonephritis, we therefore quantified c-MYC levels in in infected human kidney epithelial cells in vitro, and in kidneys from infected C57BL/6 WT mice. Both experiments showed a marked reduction in epithelial MYC levels, suggesting that the bacteria activate the cellular MYC degradation machinery and possibly suppress MYC expression. Screening of clinical isolated showed that MYC suppression was characteristic mainly of uropathogenic E. coli and not of the more commensal like ABU strains.
[0131] To identify bacterial factors responsible for c-MYC regulation, we used a systematic gene-deletion strategy, successively removing pathogenicity island genes from the E. coli 536 chromosome. MYC inhibition was regulated by genes on Pathogenicity island I and a Zn.sup.2+ -dependent ABC transporter was identified as a key regulator of the bacterial MYC-inhibitor, with partial effects of hly1. A list of potential bacterial MYC inhibitors was identified in bacterial supernatants by LC-MS/MS. The bacterial Lon protease was internalized by host cells and reduced cellular MYC levels in a dose dependent manner, possibly as a result of direct binding and proteolytic degradation of MYC. was detected in vitro one of the potential MYC-inhibitors, and by addition of recombinant Lon protease to cells, c-MYC levels were reduced, and Lon protease was showed to effectively degrade c-MYC in vitro.
[0132] Relevance to different forms of cancer was supported by a reduction in cellular MYC levels after infection or challenge with bacterial supernatants. Finally, therapeutic efficacy was documented in a murine bladder cancer model, treating tumor-bearing mice with bacterial supernatants before evaluating the treatment effects on the tumors by pathological assessment and genome-wide transcriptomic analysis.
[0133] Bacterial Strains
[0134] APN (n=36) isolates and ABU (n=20) isolates were isolated during a prospective study of childhood UTI in Gothenburg, Sweden. The prototype strains E. coli CFT073 (O6:K2:H1) (28), E. coli 536 (O6:K15:H31) and E. coli 83972 (OR:K5:H-) (31) were used as control strains. Bacteria was grown on trypsin soy agar (TSA) plates at 37° C. for 16 hours and was harvested in phosphate buffered saline (PBS, pH 7.2) and appropriately diluted for infection.
[0135] Sub-Cloning of E. coli 536 ΔPAI I
[0136] The deletion mutants of E. coli 536 has previously been described. The three mutants 536 PAI IΔ1, 536 PAI IΔ2 and 536 PAI IΔ3 as well as the subsequent mutants of 536 PAI IΔ2 were generated using lambda red homologous recombination by replacing the corresponding genomic region by a chloramphenicol acetyltransferase gene (cat) cassette. The cat cassette was flanked by FRT sites and amplified from vector pKD3 with primer pairs for the corresponding mutants. The resulting PCR products were purified and used for electroporation of competent E. coli strain 536/pKD46 cells cultivated at 30° C. in the presence of 100 mM arabinose to induce the γ, β and exo genes of the λ phage Red recombinase system. The correct exchange of each of the 536 PAI I fragments against the cat cassette was confirmed by PCR.
[0137] Bacterial Supernatants
[0138] Supernatants were prepared by growing 10.sup.8 CFU/ml bacteria in RPMI-1640 for 4 hours followed by centrifugation at 16,000 RPM for 10 minutes. Supernatants were filtered using a 0.2 μm filter before being added to cells.
[0139] Cell Culture
[0140] Human kidney carcinoma cells (A498), bladder carcinoma cells (HTB9), Burkitt lymphoma cells (Rajii and Daudi), hepatocellular carcinoma (HEP-G2), and mouse bladder cells (MB49) was cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS) Colorectal adenocarcinoma cancer (DLD1) and lung carcinoma (A549) was cultured in RPMI-1640 supplemented with 5% FCS. Gastric adenocarcinoma (AGS) was cultured in F12K medium supplemented with 10% FCS. Cells were cultured at 37° C. with 5% CO.sub.2.
[0141] In Vitro Infection, Bacterial Supernatant Treatment or Recombinant Protein Treatment
[0142] Cells were grown in 8-well chamber slides or 6-well plates overnight in appropriate media supplemented with FCS. Cells were washed, and serum free media was added prior to infection with appropriately diluted bacteria for 1 or 4 hours. For cells challenged with supernatants, cells were washed, and sterile bacterial supernatants were added to the cells for 4 hours. The challenged cells were analyzed by confocal microscopy, western blotting, qRT-PCR or whole genome transcriptomic analysis.
[0143] Divalent Ion Experiment
[0144] E. coli 536 was grown on TSA, harvested and washed with PBS. 10.sup.8 CFU/ml of bacteria was incubated with 100 μM of ZnCl.sub.2, MgCl.sub.2, MnCl.sub.2 or CaCl.sub.2) at 37° C. for 1 hour. Bacteria were then washed in PBS and appropriately diluted before adding to the cells.
[0145] Inhibitor Experiment
[0146] Cells were pre-treated with 50 μg/ml EGTA (Egtazic acid, Ca.sup.2+ chelator; Sigma) or 100 μg/ml IC261 (CK1α1 inhibitor; Sigma) for 30 minutes before bacteria infection or supernatant treatment.
[0147] Confocal Microscopy
[0148] Kidney epithelial cells were stained with rabbit anti-c-MYC (Abcam) or mouse anti-his6-tag primary antibodies (1:200 in 5% FBS/PBS, overnight at 4° C.), followed by secondary goat anti-rabbit Alexa Fluor 488-conjugated or goat-anti mouse Alexa Fluor 568-conjugated antibodies (Molecular Probes, A-11034) (1:200 in 5% FBS and 0.025% Triton X-100/PBS, 1 hour at room temperature). Cells were counterstained with DRAQ5 (Abcam) and examined using a LSM 510 META laser scanning confocal microscope (Carl Zeiss). Fluorescence signal was quantified in 50 cells per condition using Image J.
[0149] Co-Immunoprecipitation
[0150] Magnetic protein-G coupled dynabeads (Thermo Scientific) were washed in PBS, coated with antibodies directed to c-MYC (1:100, 30 minutes), incubated with recombinant c-MYC (500 ng, 30 minutes), and incubated with 50 μg of whole cell lysates, or with bacterial supernatants (1 hour). Proteins were eluted and prepared for western blot analysis.
[0151] Western Blotting
[0152] Cells were lysed with NP-40 lysis buffer supplemented with protease and phosphatase inhibitors (Roche diagnostics). Proteins were run on SDS-PAGE (4-12% Bis-Tris gels, Invitrogein), blotted onto PVDF membranes (Biorad), blocked and stained with primary rabbit anti-c-MYC (1:1000, Abcam), rabbit anti-CK1α1 (1:1000,) or rabbit-anti-Lon (1:2000, Biorbyt) overnight at 4° C. Membranes were washed and stained with HRP-conjucated goat anti-rabbit HRP conjugated secondary antibodies (1:4000, Cell Signaling) washed again, imaged using ECL plus detection reagent (GE Health Care) and quantified by ImageJ. β-actin was used as a loading control.
[0153] Whole Genome Transcriptomic Analysis
[0154] Total RNA was extracted from A498 cells using the RNeasy Mini Kit (Qiagen) and on-column DNase digestion. 100 ng of RNA was amplified using GeneChip 3′IVT PLUS Kit (ThermoFisher Scientific), then fragmented and labeled aRNA was hybridized onto Human Genome U219 or Mouse Genome MG430 array strips (16 hours at 45° C.), washed, stained and scanned in-house using the GeneAtlas system (Affymetrix). All samples passed the internal quality controls included in the array strips (signal intensity by signal to noise ratio; hybridization and labeling controls; sample quality by GAPDH signal and 3′-5′ ratio <3).
[0155] Data was normalized using Robust Multi Average implemented in the Partek Express Software (32, 33). Significantly altered genes were calculated by comparing cells treated with bacteria or bacterial supernatants to uninfected cells (PBS control) and sorted by relative expression (2-way ANOVA model using Method of Moments, absolute fold change >2.0) (34). Heat-maps were constructed using the free Gitools 2.1 software. Differentially expressed genes and regulated pathways were analyzed using the Ingenuity Pathway Analysis software (IPA, Ingenuity Systems, Qiagen).
[0156] Quantitative Real-Time (qRT)-PCR
[0157] Total RNA was reverse-transcribed using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) and quantified in real time using QuantiTect SYBR Green-based PCR Kits for mRNA (QIAGEN) on a RotorGene Q cycler (QIAGEN). Human specific primers were described in Table 3. Quantitative RT-PCR (qRT-PCR) reactions were run in biological duplicates and triplicates, and gene expression was quantified using the 2.sup.−ΔΔCT method.
[0158] In-Gel Digestion and Mass Spectrometry
[0159] TCA precipitated supernatants from E. coli 536 and ΔPAI I was loaded on a NU-PAGE gel and stained with Coomassie blue. Five bands were excised from the E. coli 536 lane and five bands corresponding in size in the ΔPAI I lane. Bands were digested in trypsin followed by identification using liquid chromatography-tandem mass-spectrometry (LC-MS/MS). Analysis was performed on a tibrid mass spectrometer fusion (Thermo Fischer Scientific). Samples were injected and concentrated onto an Acclaim PepMap 100 C18 precolumn (75 μm×2 cm, Thermo Scientific, Waltham, Mass.) and separated on a Acclaim PepMap RSLC column (75 μm×25 cm, nanoViper, C18, 2 μm, 100 Å) at 40° C. Separation was performed at a flow rate of 300 nl/min in 135 min with a non-linear gradient ranging from 3% to 90% acetonitrile containing 0.1% formic acid. Raw data were analysed with Proteome Discoverer version 2.2 (Thermo Fisher Scientific). Peptides were identified using SEQUEST HT against UniProtKB EColi database (release 2018 Dec. 6). Precursor tolerance was set to 10 ppm and fragment tolerance was set to 0.05 ppm. Up to 2 missed cleavages were allowed and Percolator was used for peptide validation at a q-value of maximum 0.01.
[0160] Murine Urinary Tract Infection Model
[0161] Female C57BL/6 WT mice were used for experiments at 9-15 weeks of age, weighing 21-26 g. The mice were bred and housed in the specific pathogen-free MIG animal facilities (Lund, Sweden) with free access to food and water. Mice were intravesically infected with E. coli CFT073 or E. coli 536 under Isofluorane anesthesia (10.sup.8 CFU in 0.1 ml), through a soft polyethylene catheter (outer diameter 0.61 mm; Clay Adams). Animals were sacrificed under anesthesia by cervical dislocation at 24 hours and bladders were aseptically removed and frozen into cryomolds containing O.C.T. Urine counts were determined by growth on TSA plates (37° C., overnight) after serial dilutions. Neutrophils from uncentrifuged urine were quantified using a hemocytometer.
[0162] Bladder Cancer Model
[0163] C57BL/6 female mice were bred at the Department of Laboratory Medicine and used at ages from 7 to 12 weeks. For procedures, mice were anesthetized by intraperitoneal injection of a ketamine and xylazine cocktail. MB49 tumors were established as described (25). On day 0, the bladder was emptied and preconditioned by intravesical instillation of 100 μl poly-L-lysine solution (0.1 mg/ml) through a soft polyethylene catheter (Clay Adams, Parsippany, N.J.) with an outer diameter of 0.61 mm for 30 minutes before MB49 tumor cells (2×10.sup.5 in 100 μl PBS) were instilled. On days 3, 5, 7, 9 and 11, 100 μl of E. coli 536 or ΔPAI I bacterial supernatant or PBS (sham-treated controls) were instilled. Mice remained under anesthesia on preheated blocks with the catheter in place to prolong tumor exposure to E. coli 536 or ΔPAI I bacterial supernatant (approximately 1 hour). Groups of 5-7 mice for each treatment and control were sacrificed at each time point, and bladders were imaged and processed for histology.
[0164] Histology and Immunohistochemistry
[0165] Bladders were embedded in O.C.T. compound (VWR), and 5-μm-thick fresh cryosections on positively-charged microscope slides (Superfrost/Plus; Thermo Fisher Scientific) were fixed with 4% paraformaldehyde or acetone-methanol (1:1 v/v). For hemotoxylin-eosin (H&E) staining, Richard-Allan Scientific Signature Series Hematoxylin 7211 and Eosin-Y 7111 (Thermo Fisher Scientific) were used to counterstain the tissue sections. Imaging was done with AX10 (Carl Zeiss). Cryosections were permeabilized (0.25% Triton X-100, 5% fetal calf serum/PBS) and incubated with primary anti-c-MYC antibody (1:500, 4° C., overnight, ab39688, Abcam). This was followed by staining with Alexa Fluor® 488-labeled secondary goat anti-rabbit antibodies (1:200, 1 hour, room temperature, A-11011, Molecular Probes). Tissues were counterstained with (1:1000, 15 minutes, room temperature, Abcam) and examined using an LSM 510 confocal microscope (Carl Zeiss). The quantification of images and fluorescence was performed in ImageJ.
[0166] Clinical Pilot Study of UTI Prone Patients
[0167] To characterize gene expression levels in UTI prone patients during an infection free interval, we sequenced peripheral blood RNA from 5 children with a history of acute pyelonephritis and x children with long-term ABU. RNA was obtained during an infection-free interval, a least one year after the last episode of acute pyelonephritis or a positive urine culture in the ABU group. The patients were followed for at least six years and their susceptibility pattern and history of infection was documented by a pediatric nephrologist (D Karpmann), at the Department of Pediatrics, Lund University Hospital. A diagnosis of APN was based on fever (>38.5° C.) with significant bacteriuria, C-reactive protein (CRP)>20 mg/I and no symptoms of other infections. A diagnosis of ABU was based on at least three consecutive urine cultures yielding the same bacterial strain (>10.sup.5 CFU/ml of urine) in a child with no symptoms of UTI and no CRP increase. The APN group had not developed ABU at any time during follow-up. Informed written consent was obtained from all participants or their parents/guardians. The study was approved by the Ethics Committee of the medical faculty, Lund University, Sweden (LU106-02, LU236-99).
[0168] RNA Sequencing
[0169] RNA was extracted using the QUiamp RNA blood mini kit (Qiagen) or the PAXgene™ Blood RNA tube and PAX gene blood RNA system. Quantity and quality was determined by Nanodrop and bioanalyzer before proceeding with RNA-Seq library preparation. The quality of the library was evaluated using the Agilent Technologies 2100 Bioanalyzer and a DNA 1000-kit. The quantity of adapter ligated fragments was determined by qPCR using the KAPA SYBR FAST library quantification kit for Illumina GA (KAPA Biosystems). A 10 μM solution of the sequencing library was used in the cluster generation on the Cluster Station (Illumina Inc.). Paired-end sequencing with 100 bases read length was performed using the Genome Analyzer.sub.IIx, according to the manufacturer's protocols. Sequence reads were mapped using Tophat using default settings and reads were aligned to human reference genome Hg37.2.
[0170] Bioinformatic Analysis
[0171] Log.sub.2 FC<0.8 was used as cut-off for bioinformatic analysis. Functional changes and activated canonical pathways were analyzed with using Ingenuity Pathways Analysis (IPA, Qiagen). Gene Set Enrichment Analysis (GSEA, Broad institute, Cambride, Mass.) was used to assess whether a defined set of genes showed statistically significant, concordant differences between the patient groups.
[0172] Quantification and Statistical Analysis
[0173] For data determined to follow a Gaussian distribution (D'Agostino & Pearson normality test), statistical differences were determined by two-tailed t-test or one-way ANOVA. For non-parametric data, Mann-Whitney U-tests and Kruskal-Wallis tests (Dunn's correction) was used. Statistical significance was determined by GraphPad (Prism v. 7) and significance was assigned at *P<0.05, **P<0.01 and ***P<0.001.
TABLE-US-00001 TABLE 1 Top 50 up-regulated transcripts in APN patients (⅔) log2 fold-change Genes Entrez Gene Name APN6 APN7 APN10 Immune responses/ T cell signaling or function DPP4 Dipeptidyl-peptidase 4 8.55 8.08 3.88 CD8A CD8a molecule 7.96 7.13 3.95 CD28 CD28 molecule 6.86 7.33 3.94 CD226 CD226 molecule 7.21 7.41 3.43 CD247 CD247 molecule 7.10 7.22 3.51 CCR7 Chemokine (C—C motif) receptor 7 6.57 6.33 4.70 GZMK Granzyme K (granzyme 3; tryptase II) 5.82 5.72 5.78 PRF1 Perforin 1 (pore forming protein) 7.09 5.72 4.22 IL7R Interleukin 7 receptor 6.38 5.65 4.98 GZMB Granzyme B (granzyme 2, cytotoxic T- 6.62 5.70 4.61 lymphocyte-associated serine esterase 1) CD3D CD3d molecule, delta (CD3-TCR complex) 6.26 6.05 4.39 Immune responses/ B cell signaling or function CD72 CD72 molecule 7.62 7.38 2.72 FCRLA Fc receptor-like A 7.32 6.92 3.40 BLK B lymphoid tyrosine kinase 6.93 6.40 3.92 VPREB3 Pre-B lymphocyte 3 6.67 6.93 3.53 TNFRSF13C Tumor necrosis factor receptor superfamily, 6.45 6.39 4.25 member 13C FCRL5 Fc receptor-like 5 6.58 7.01 3.33 CD79A CD79a molecule, immunoglobulin-associated 5.97 5.99 4.67 alpha Immune responses/ Other immune cells MMD Monocyte to macrophage differentiation- 8.38 8.07 2.38 associated LILRA4 Leukocyte immunoglobulin-like receptor, 7.30 6.44 4.55 subfamily A (with TM domain), member 4 PPBP Pro-platelet basic protein (chemokine 7.88 8.19 2.19 (C—X—C motif) ligand 7) KLRB1 Killer cell lectin-like receptor subfamily B, 6.40 5.48 4.79 member 1 Transcriptional regulation MYC v-myc myelocytomatosis viral oncogene 7.09 6.83 5.16 homolog (avian) SPIB Spi-B transcription factor (Spi-1/PU.1 related) 7.22 7.25 4.41 BACH 2 BTB and CNC homology 1, basic leucine 7.18 6.83 3.87 zipper transcription factor 2 PAX5 Paired box 5 6.89 6.53 4.05 HABP4 Hyaluronan binding protein 4 6.74 6.83 3.37 HNRNPA1 Heterogeneous nuclear ribonucleoprotein A1 6.50 6.15 4.44 MAF v-maf musculoaponeurotic fibrosarcoma 6.85 7.32 2.73 oncogene homolog (avian) ZNF202 Zinc finger protein 202 6.81 6.68 3.23 Cell growth and proliferation, cellular organization, cell-cell interactions NOG Noggin 6.50 7.19 6.32 VSIG1 V-set and immunoglobulin domain containing 1 6.47 6.57 5.29 GRAP GRB2-related adaptor protein 7.19 7.40 3.73 GPA33 Glycoprotein A33 (transmembrane) 6.80 7.01 4.32 SDPR Serum deprivation response 7.46 7.30 2.50 MYO IE Myosin IE 7.30 6.77 3.00 Biosynthesis/metabolism EPHX2 Epoxide hydrolase 2, cytoplasmic 7.61 7.24 3.81 CYP2U1 Cytochrome P450, family 2, subfamily U, 6.61 6.54 3.56 polypeptide 1 Membrane functions a) Lipid metabolism/receptors OSBPL10 Oxysterol binding protein-like 10 7.78 7.73 4.32 b) ion channels CLIC3 Chloride intracellular channel 3 7.86 6.69 4.42 Enzymes/ Posttranslational modifications NT5E 5′-nucleotidase, ecto (CD73) 7.08 6.71 5.77 DPH1 DPH1 homolog (S. cerevisiae) 7.59 6.78 3.69 GIMAP7 GTPase, IMAP family member 7 6.49 6.65 4.44 TBC1D9 TBC1 domain family, member 9 (with GRAM 8.14 7.44 1.36 domain) Unknown functions SPATA20 Spermatogenesis associated 20 6.80 5.83 5.60 SAMD3 Sterile alpha motif domain containing 3 6.66 6.06 5.15 OAF OAF homolog (Drosophila) 7.73 7.78 2.11 MTSS1 Metastasis suppressor 1 7.38 7.07 2.86 RPUSD2 RNA pseudouridylate synthase domain 6.70 6.35 4.07 containing 2 C12orf57 Chromosome 12 open reading frame 57 6.46 6.60 4.05 TRIM 58 Tripartite motif containing 58 7.50 6.98 2.30
TABLE-US-00002 TABLE 2 Transcription regulators activated in APN patients APN6 APN7 APN10 Transcription Regulation p-value of Regulation p-value of Regulation p-value of Regulator z-score overlap z-score overlap z-score overlap MYCN 4.703 5.66E−13 4.575 3.85E−13 5.127 1.16E−15 MYC 4.423 9.19E−04 3.934 3.77E−04 4.087 5.45E−09 KLF2 3.128 2.11E−01 2.599 3.69E−01 3.527 4.10E−01 NCOR2 2.033 2.78E−01 — 2.029 6.25E−02 WT1 2.209 9.06E−02 — — — ESR1 2.006 5.13E−01 — — — CIITA — — 2.31 1.41E−01 — CUX1 — — 2.068 1.06E−01 — —
TABLE-US-00003 TABLE 3 qRT-PCR primers Gene Primer sequence c-MYB Fwd 5′-ATTGTGGACCAGACCAGACC-3′ Rev 5′-GCTGGTGAGGCACTTTCTTC-3′ c-MYC QuantiTect c-MYC Primer Assay (Qiagen catalog # QT00035406) CSNK1A1 Fwd 5′-ATGGCGAGTAGCAGCGGCTC-3′ Rev 5′-CGTATGTGGGGGATGCCAAC-3′ CEBPD Fwd 5′-AGAAGTTGGTGGAGCTGTCG-3′ Rev 5′-CAGCTGCTTGAAGAACTGCC-3′ MAX Fwd 5′-GTTTCAATCTGCGGCTGACA-3′ Rev 5′-GAAGAGCATTCTGCCGCTTG-3′ GAPDH QuantiTect GAPDH Primer Assay (Qiagen catalog # QT00079247)
Example 5
[0174] Rapidly c-MYC Depletion in Response to Infection
[0175] Epithelial cells are targeted when bacteria ascend into the renal pelvis and cause acute pyelonephritis. While investigating molecular mechanisms of this disease, we discovered that epithelial c-MYC levels are modified by bacterial infection. A rapid reduction in c-MYC protein levels was detected following in vitro infection with uropathogenic E. coli (CFT073 and 536) (
[0176] c-MYC Inhibition
[0177] To address the human relevance of these findings, MYC expression was quantified in a population-based study of febrile urinary tract infection in infants and young children. A diagnosis of acute pyelonephritis was based on a positive urine culture, a temperature >38° C. and a positive DMSA scan, confirming renal involvement. RNA was obtained from peripheral blood at the time of diagnosis and six months later, at the follow up visit after the completion of antibiotic therapy (
[0178] Gene Deletion Strategy to Identify Bacterial c-MYC Regulators/Inhibitors
[0179] To identify mechanism(s) of bacterial c-MYC inhibition, we selected the uropathogenic strain E. coli 536.sup.14,15, in which virulence genes and chromosomal pathogenicity islands (PAIs) have been extensively characterized (
[0180] Sequential deletions from the 5′ end of PAI I were generated using lambda red homologous recombination.sup.20 (
[0181] Mechanism(s) of MYC Degradation in Host Cells
[0182] Transcription factors are rapidly degraded in host cells and c-MYC has an estimated half-life of around 30 minutes.sup.17. The reduction in c-MYC protein levels suggested that bacterial effector molecules may engage and degrade c-MYC or accelerate endogenous mechanisms of c-MYC protein degradation. To identify bacterial effectors of MYC inhibition, bacterial supernatants were subjected to mass spectrometry. The Lon protease was identified as an abundant constituent of active supernatants from E. coli 536 compared to inactive supernatants from E. coli ΔPAI I.sub.536 (
[0183] The bacterial Lon protease has been shown to target serine rich proteins under conditions of stress, suggesting that a high serine content may define the affinity of Lon for c-MYC (serine content 14.3%). A direct interaction of c-MYC with recombinant Lon protease was verified by co-immunoprecipitation of E. coli 536 supernatants, using c-MYC-coated Protein G-coupled dynabeads. Supernatants from E. coli 536 Δlon were negative (
[0184] Uptake of the recombinant Lon protease by human kidney cells was detected by confocal microscopy and the increase in cytoplasmic Lon staining was accompanied by a dose-dependent decrease in c-MYC staining (
[0185] Weak pull-down of recombinant MAX (MYC associated factor X) was observed but degradation of MAX by Lon was not detected in vitro. Upstream regulators of c-MYC such as AKT, CDK9, CBEB1 were not degraded by Lon in vitro nor were housekeeping proteins HH3, Actin and GRP75 (
[0186] Serine content (% total amino acids) of proteins in
TABLE-US-00004 TABLE 4 Protein Serine content (%) c-MYC 14.4% MAX 14% C/EBP-δ 6% C-MYB 8.5% AKT 4.8% CDK9 5.1% CPEB1 12.9% GRP75 6% Actin 6% HH3 3.5%
[0187] Hly Dependent Activation of CK1α1 and MYC Degradation
[0188] A second mechanism of c-MYC degradation was identified by gene expression analysis of infected cells. The kinase CK1α1 phosphorylates c-MYC at Serine.sup.252, marking the protein for proteasomal degradation.sup.18. E. coli 536 increased CSNK1A1 expression and the kinase was activated in infected cells. By co-immunoprecipitation, binding of c-MYC to CK1α1 was detected in infected cell lysates and pharmacologic inhibition of CK1α1 with IC261 reduced c-MYC degradation.sup.19, linking CK1α1 activation to MYC degradation (
[0189] The results suggest that c-MYC is degraded in infected cells by direct Lon protease cleavage and by an alpha-hemolysin induced loop of CK1α1 activation.
[0190] Bacterial Inhibition of MYC Expression
[0191] c-MYC works in concert with a large number of transcriptional regulators and c-MYB acts as a transcriptional enhancer, trans-activating the MYC promoter by binding distal upstream regulatory regions.sup.23,24. Transcriptomic analysis identified a network of differentially regulated MYB-related genes in infected cells (E. coli 536 vs. PAI I.sub.536
[0192] The results suggest that in addition to accelerating c-MYC degradation, bacteria inhibit c-MYC expression by targeting transcriptional c-MYC regulators. The identified mechanisms of c-MYC degradation/inhibition are summarized in
[0193] Broad c-MYC Suppression in Cancer Cells from Different Tissues
[0194] The MYC family of oncogenes is subject to “a bewildering assortment of genetic rearrangements” associated with many different tumors cell types.sup.27. To understand if bacterial c-MYC suppression is generally relevant, tumor cell lines from different tissues were screened. E. coli 536 infection reduced cellular c-MYC levels in Burkitt lymphoma cells, where c-MYC was first discovered.sup.17, as well as gastric (AGS) and hepatic (HEP-G2) carcinoma cells (
[0195] Therapeutic Efficacy of the Lon Protease
[0196] Therapeutic efficacy of the bacterial c-MYC inhibitors was first examined in a murine bladder cancer model. Bladder cancer is common and among the most costly cancer forms, due to high recurrence rates and a lack of curative therapies.sup.28,29,30,36. Bladder cancer was established in C57BL/6 mice by intra-vesical instillation of rapidly growing MB49 tumor cells (day 0,
[0197] Therapeutic efficacy of the Lon protease was further demonstrated in the murine Apc.sup.Min/+ model of colon cancer. The model mimics human disease, especially familial polyposis where APC mutations increase the colon cancer susceptibility.sup.31. The treatment groups received recombinant Lon protease by oral gavage, twice daily for 14 days and the sham group received PBS (
[0198] Long-Term Follow Up and Toxicity
[0199] Long-term efficacy of Lon treatment was further investigated by follow up after the end of treatment on day 12, when the sham group was sacrificed. Lon treated mice remained asymptomatic until day 27, when hematuria was detected in 3/15 mice and the group was sacrificed. Bladder tumors were present in 15/15 mice but bladder weights and pathology scores remained lower than in the sham group (
[0200] The APC.sup.Min+/− mice were also followed long-term after the end of treatment, without further intervention. The sham group developed symptoms on days 119-175 (weight loss, restricted movement) but the Lon treated mice remained asymptomatic until day 175 (survival curve in
[0201] Three approaches were taken to address if the Lon protease causes significant toxicity. First, the Lon concentrations that inhibited c-MYC in vitro were not found to reduce human kidney cell viability, suggesting that the decrease in c-MYC levels is not due to cell death (
TABLE-US-00005 TABLE 5 Comparison of E. coli 536 supernatant and rLon with classical bladder cancer therapeutics Bladder size Bladder weight Treatment % Sham (range) g .sup.−1 (range) Sham 100 (61-149).sup.a 10.7 (8.2-13.4).sup.a n = 17 536 SN 54 (43-68).sup.a 5.5 (3.9-6.6).sup.a n = 14 rLon 69 (53-85).sup.a 5.3 (3.9-6.3).sup.a n = 7 Mitomycin 67 (54-87).sup.b 4 (3.4-5.9).sup.b 25 μg n = 6 Epirubicin 65 (53-70).sup.b 3.9 (3.2-4.8).sup.b 25 μg n = 6
[0202] Materials and Methods for Example 5
[0203] Patients
[0204] Total RNA from peripheral blood was obtained from a prospective analytical cohort study at the KK Woman and Children's hospital in Singapore. Patients were enrolled randomly after written consent was taken in accordance to requirements of Institution ethics committee. The enrolled patients were children aged >1 months with a clinical UTI diagnosis based on fever without known source, pyuria and bacteriuria (>10.sup.4 CFU/ml for catheterized urine, or >10.sup.5 CFU/ml in mid-stream clean-catch urine). Exclusion criteria were neonates and those with atypical presentation of recurrent UTI, underlying renal or urological abnormalities. Peripheral blood samples were taken at the time of infection and at a six-month follow up visit.
[0205] Transcriptome and Pathway Analysis
[0206] The transcriptomic profile was analyzed in a subset of the enrolled patients with confirmed APN (n=18). Total RNA was purified from peripheral blood collected in Tempus blood RNA tubes (Applied Biosystems) and subjected to gene expression measurements using Affymetrix GeneChip Human Gene 2.1 ST Arrays. All arrays were quality controlled by using Normalized Unscaled Standard Errors (NUSE.sup.41). Fold Change was calculated by comparing intraindividual samples during APN and at six-month follow-up. Significantly altered genes were sorted by relative expression (2-way ANOVA model using Method of Moments, P-values <0.05 and absolute fold change >1.41) and analyzed using gene set enrichment analysis (GSEA).
[0207] Quantitative Real-Time (qRT)-PCR
[0208] Total RNA was reverse-transcribed using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) and quantified in real time using QuantiTect SYBR Green-based PCR Kits for mRNA (QIAGEN) on a RotorGene Q cycler (QIAGEN). Human specific primers are described in table 7 below. Quantitative RT-PCR (qRT-PCR) reactions were run in biological duplicates.
TABLE-US-00006 TABLE 6 qRT-PCR primers Gene Primer sequence MYB Fwd 5′-ATTGTGGACCAGACCAGACC-3′ Rev 5′-GCTGGTGAGGCACTTTCTTC-3′ MYC QuantiTect c-MYC Primer Assay (Qiagen catalog # QT00035406) CSNK1A1 Fwd 5′-ATGGCGAGTAGCAGCGGCTC-3′ Rev 5′-CGTATGTGGGGGATGCCAAC-3′ CEBPD Fwd 5′-AGAAGTTGGTGGAGCTGTCG-3′ Rev 5′-CAGCTGCTTGAAGAACTGCC-3′ MAX Fwd 5′-GTTTCAATCTGCGGCTGACA-3′ Rev 5′-GAAGAGCATTCTGCCGCTTG-3′ GAPDH QuantiTect GAPDH Primer Assay (Qiagen catalog # QT00079247)
[0209] Bacterial Strains
[0210] APN (n=36) isolates and ABU (n=20) isolates were isolated during a prospective study of childhood UTI in Gothenburg, Sweden.sup.12,13. The prototype strains E. coli CFT073 (O6:K2:H1).sup.42, E. coli 536 (O6:K15:H31).sup.15 and E. coli 83972 (OR:K5:H-).sup.43 were used as control strains. Bacteria was grown on trypsin soy agar (TSA) plates at 37° C. for 16 hours and was harvested in phosphate buffered saline (PBS, pH 7.2) and appropriately diluted for infection.
[0211] Sub-Cloning of E. coli ΔPAI I.sub.536
[0212] The complete PAI deletion mutants of E. coli strain 536 have previously been described.sup.44. The three mutants PAI I.sub.536Δ1, PAI I.sub.536Δ2 and PAI I.sub.536Δ3 which lack different regions of PAI I.sub.536 as well as the subsequent deletion of selected genes included within region 2 of PAI I.sub.536 were generated using lambda Red-based homologous recombination by replacing the corresponding genomic region or genes by a chloramphenicol acetyltransferase gene (cat) cassette. The cat cassette was flanked by FRT sites and amplified from vector pKD3 with primer pairs for the corresponding mutants. The resulting PCR products were purified and used for electroporation of competent E. coli strain 536/pKD46 cells cultivated at 30° C. in the presence of 100 mM arabinose to induce the γ, β and exo genes of the λ phage Red recombinase system. The correct exchange of each of the PAI I.sub.536 fragments against the cat cassette was confirmed by PCR. Deleted genes and ORFs are shown in the following table.
TABLE-US-00007 TABLE 7 Deleted genes/ORFs encoded on PAI I.sub.536. (Supplementary information for FIG. 16). PAI I Gene/ deletion ORF's ORF mutant * Deleted designation Function 1 ECP_3765- ECP_3796 2 ECP_3798- ECP_3829 3 ECP_3830- ECP_3863 2-1 ECP_3798- ECP_3798 Inovirus GP2 family protein ECP_3801 ECP_3799 Hypothetical protein ECP_3800 AlpA family transcriptional regulator ECP_3801 Hypothetical protein 2-2 ECP_3802- ECP_3804 IS110 family transposase (frame shifted) ECP_3809 ECP_3805 IS110 family transposase (frame shifted) ECP_3806 IS3 family transposase (frame shifted) ECP_3807 IS3 family transposase (frame shifted) ECP_3808 IS4 like element ISSfl9 family transposase (frame shifted) ECP_3809 Hypothetical protein 2-3 ECP_3810- ECP_3810 AraC family transcriptional regulator ECP_3817 ECP_3811 CS12 fimbriae major subunit ECP_3812 CS12 fimbriae periplasmic chaperone ECP_3813 CS12 fimbriae chaperone protein ECP_3814 CS12 outer membrane usher protein ECP_3815 CS12 fimbriae chaperone protein ECP_3816 CS12 fimbriae minor subunit protein precursor ECP_3817 CS12 fimbriae adhesin protein 2-4 ECP_3818- ECP_3818 IS110 family transposase ECP_3821 ECP_3819 Hypothetical proten ECP_3820 Helix-turn-helix domain containing protein ECP_3821 Hypothetical protein 2-5 ECP_3821 ECP_3821 Hypothetical protein (partial)- ECP_3822 Putative ABC metal-type transporter ATP-binding protein ECP3825 ECP_3823 Putative ABC metal-type transporter permease ECP_3824 Putative ABC metal-type substrate binding protein ECP_3825 Hypothetical protein hly I.sup.§ ECP_3826- ECP_3826 Alpha-hemolysin-activating lysine-acyltransferase HlyC ECP3829 ECP_3827 RTX toxin alpha-hemolysin HlyA ECP_3828 Type I secretion system permease/ATPase HlyB ECP_3829 Alpha-hemolysin TISS ABC transporter subunit HlyD *Mutants in FIG. 16e-g .sup.§Mutant with partial effect in FIG. 16d.
[0213] Bacterial Supernatants
[0214] Supernatants were prepared by growing 10.sup.8 CFU/ml bacteria in RPMI-1640 for 4 hours followed by centrifugation at 16,000 RPM for 10 minutes. Supernatants were filtered using a 0.2 μm filter before being added to cells.
[0215] Cell Culture
[0216] Human kidney carcinoma cells (A498), bladder carcinoma cells (HTB9), Burkitt lymphoma cells (Rajii and Daudi), hepatocellular carcinoma (HEP-G2), and mouse bladder cells (MB49) was cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS) Colorectal adenocarcinoma cancer (DLD1) and lung carcinoma (A549) was cultured in RPMI-1640 supplemented with 5% FCS. Gastric adenocarcinoma (AGS) was cultured in F12K medium supplemented with 10% FCS. Cells were cultured at 37° C. with 5% CO.sub.2.
[0217] In Vitro Infection, Bacterial Supernatant Treatment or Recombinant Protein Treatment
[0218] Cells were grown in 8-well chamber slides or 6-well plates overnight in appropriate media supplemented with FCS. Cells were washed, and serum free media was added prior to infection with appropriately diluted bacteria for 1 or 4 hours. For cells challenged with supernatants, cells were washed, and sterile bacterial supernatants were added to the cells for 4 hours. The challenged cells were analyzed by confocal microscopy, Western blotting, qRT-PCR or whole genome transcriptomic analysis.
[0219] Divalent Ion Experiment
[0220] E. coli 536 was grown on TSA, harvested and washed with PBS. 10.sup.8 CFU/ml of bacteria was incubated with 100 μM of ZnCl.sub.2, MgCl.sub.2, MnCl.sub.2 or CaCl.sub.2) at 37° C. for 1 hour. Bacteria were then washed in PBS and appropriately diluted before adding to the cells.
[0221] Inhibitor Experiment
[0222] Cells were pre-treated with 50 μg/ml EGTA (Egtazic acid, Ca.sup.2+ chelator; Sigma) or 100 μg/ml IC261 (CK1α1 inhibitor; Sigma) for 30 minutes before bacterial infection or supernatant treatment.
[0223] Confocal Microscopy
[0224] Kidney epithelial cells were stained with rabbit anti-c-MYC (Abcam), rabbit anti-CK1α1 (Cell Signaling Technology), rabbit anti-c-MYB (Sigma Aldrich), rabbit anti-CEBPD (Abcam) or mouse anti-his6-tag primary antibodies (1:200 in 5% FBS/PBS, overnight at 4° C.), followed by secondary goat anti-rabbit Alexa Fluor 488-conjugated or goat-anti mouse Alexa Fluor 568-conjugated antibodies (Molecular Probes, A-11034) (1:200 in 5% FBS and 0.025% Triton X-100/PBS, 1 hour at room temperature). Cells were counterstained with DRAQ5 (Abcam) and examined using a LSM 510 META laser scanning confocal microscope (Carl Zeiss). Fluorescence signal was quantified in 50 cells per condition using Image J.
[0225] Co-Immunoprecipitation
[0226] Magnetic protein-G coupled dynabeads (Thermo Scientific) were washed in PBS, coated with antibodies directed to GST or c-MYC (1:100, 30 minutes), incubated with recombinant c-MYC or MAX protein (500 ng, 30 minutes), and incubated with whole cell lysates (50 μg), bacterial supernatants, or recombinant Lon protease (0.2 μg) for 1 hour. Proteins were eluted and prepared for western blot analysis.
[0227] Western Blotting
[0228] Cells were lysed with NP-40 lysis buffer supplemented with protease and phosphatase inhibitors (Roche diagnostics). Proteins were run on SDS-PAGE (4-12% Bis-Tris gels, Invitrogen), blotted onto PVDF membranes (Biorad), blocked and stained with primary rabbit anti-c-MYC (1:1000, Abcam), rabbit anti-CK1α1 (1:1000, Cell Signaling Technology) or rabbit-anti-Lon (1:2000, Biorbyt) overnight at 4° C. Membranes were washed and stained with HRP-conjugated goat anti-rabbit HRP conjugated secondary antibodies (1:4000, Cell Signaling) washed again, imaged using ECL plus detection reagent (GE Healthcare) and quantified by ImageJ. β-actin was used as a loading control.
[0229] Cell Toxicity Assays
[0230] Cells (4×10.sup.4 cells/well) were seeded in serum-free RPMI-1640 on 96-well plates (Tecan Group Ltd). Cells were treated with recombinant Lon protease and incubated for four hours. The cell viability or cell death was quantified by three different assays. The released ATP was determined by the luminescence-based ATP Lite™ kit (Perkin Elmer). The mitochondrial enzymes activity was determined by the Presto Blue assay (Invitrogen, A13262). The lactate dehydrogenase (LDH) released from damaged cells were quantified by LDH assay (ThermoScientific, 88953). Assays were repeated twice in triplicate and mean value was calculated.
[0231] In-Gel Digestion and Mass Spectrometry
[0232] TCA precipitated supernatants from E. coli 536 and PAI I.sub.536 was loaded on a NU-PAGE gel and stained with Coomassie blue. Five bands were excised from the E. coli 536 lane and five bands corresponding in size in the E. coli PAI I.sub.536 lane. Bands were digested in trypsin followed by identification using liquid chromatography-tandem mass-spectrometry (LC-MS/MS). Analysis was performed on a tibrid mass spectrometer fusion (Thermo Fischer Scientific). Samples were injected and concentrated onto an Acclaim PepMap 100 C18 precolumn (75 μm×2 cm, Thermo Scientific, Waltham, Mass.) and separated on a Acclaim PepMap RSLC column (75 μm×25 cm, nanoViper, C18, 2 μm, 100 Å) at 40° C. Separation was performed at a flow rate of 300 nl/minute in 135 minutes with a non-linear gradient ranging from 3% to 90% acetonitrile containing 0.1% formic acid. Raw data were analyzed with Proteome Discoverer version 2.2 (Thermo Fisher Scientific). Peptides were identified using SEQUEST HT against UniProtKB EColi database (release 2018 Dec. 6). Precursor tolerance was set to 10 ppm and fragment tolerance was set to 0.05 ppm. Up to 2 missed cleavages were allowed and Percolator was used for peptide validation at a q-value of maximum 0.01.
[0233] In Vitro Degradation Assay
[0234] 0.1 μg of recombinant proteins were incubated with 0.2 μg of Lon protease (Abcam, ab141921), or PBS, in a reaction buffer consisting of 20 mM MgCl.sub.2 in the presence of ATP (2 mM) in 50 mM Tris. pH 8.0. Proteins were incubated at 37° C. for 60, 120 or 240 minutes and were denatured using 10 mM DTT in NuPAGE Sample Buffer at 100° C. for 5 minutes before further analysis.
[0235] Identification of c-MYC Cleavage by Lon Protease
[0236] Recombinant c-MYC protein 1 μg was incubated with 0.2 μg Lon protease or PBS for 4 hours at 37° C. Samples were diluted with NuPAGE LDS sample buffer (Invitrogen) mixed with 10 mM dithiotreitol (DTT) and incubated at 56° C. for 20 min followed by addition of iodoacetamide (IAA) to a final concentration of 20 mM. Samples were loaded on 4-12% NuPAGE (Invitrogen). The electrophoresis was run in MOPS buffer. The gel was Coomassie stained and bands of interest were excised from the SDS-PAGE and trypsin digested, followed by identification using LC-MS/MS as per above. MS/MS spectra were investigated with PEAKS (version 10) against UniProt Human (released 20200221) where the sequence for GST-tag-MYC had been added. The precursor tolerance and fragment tolerance were set to 10 ppm and 0.05 Da, respectively. None enzyme was selected, methionine oxidation and deamidation of asparagine and glutamine were treated as dynamic modification and carbamidomethylation of cysteine as a fixed modification.
[0237] Whole Genome Transcriptomic Analysis
[0238] Total RNA was extracted from A498 cells or whole bladder or intestinal tissue from mice using the RNeasy Mini Kit (Qiagen) and on-column DNase digestion. 100 ng of RNA was amplified using GeneChip 3′IVT PLUS Kit (ThermoFisher Scientific), then fragmented and labeled aRNA was hybridized onto Human Genome U219 or Mouse Genome MG430 array strips (16 hours at 45° C.), washed, stained and scanned in-house using the GeneAtlas system (Affymetrix). All samples passed the internal quality controls included in the array strips (signal intensity by signal to noise ratio; hybridization and labeling controls; sample quality by GAPDH signal and 3′-5′ ratio <3). Data was normalized using Robust Multi Average implemented in the Partek Express Software.sup.45,46. Significantly altered genes were calculated by comparing cells or tissue infected with bacteria to uninfected controls (PBS control) and sorted by relative expression (2-way ANOVA model using Method of Moments, absolute fold change >2.0).sup.47.
[0239] Bladder Cancer Model
[0240] C57BL/6J female mice were bred at the Department of Laboratory Medicine and used at ages from 7 to 12 weeks. For procedures, mice were anesthetized by intraperitoneal injection of a ketamine and xylazine cocktail. MB49 tumors were established as described.sup.48. On day 0, the bladder was emptied and preconditioned by intravesical instillation of 100 μl poly-L-lysine solution (0.1 mg/ml) through a soft polyethylene catheter (Clay Adams, Parsippany, N.J.) with an outer diameter of 0.61 mm for 30 minutes before MB49 tumor cells (2×10.sup.5 in 100 μl PBS) were instilled. On days 3, 5, 7, 9 and 11, 100 μl of E. coli 536 or PAI I.sub.536 bacterial supernatant, Lon protease (250 μg/ml) or PBS (sham controls) were instilled. Mice remained under anesthesia on preheated blocks with the catheter in place to prolong tumor exposure to E. coli 536 or PAI I.sub.536 bacterial supernatant (approximately 1 hour). Lon protease with an addition of a 6×HIS-tag, was commercially produced in the E. coli K12 strain with endotoxin levels <1.0 EU/μg. Groups of 5-7 mice for each treatment and control were sacrificed at each time point, and bladders were imaged and processed for histology. For long-term follow up experiments, mice were treated for 12 days with Lon protease (250 μg/ml) or received PBS (Sham) and were left until they developed signs of hematuria. Alternatively, Lon treatment (250 μg/ml) was administered twice weekly until mice developed signs of hematuria.
[0241] Colon Cancer Model
[0242] C57BL/6J-APC.sup.Min/J that develop spontaneous intestinal adenoma formation.sup.31 were bred at the Department of Laboratory medicine and used at either the age of 11 weeks, or 20 weeks. The mice were treated twice daily for 14 days using oral gavage (200 μl) of E. coli 536 WT supernatants (10 μg/kg) or using recombinant Lon Protease (250 μg/ml). At sacrifice, intestines were aseptically harvested and opened longitudinally, the number of polyps were counted in a blinded manner by two independent researchers. Different segments of the intestine were then prepared for either Swizz-rolls, protein extraction or mRNA extraction for further analysis. For long-term follow-up experiments, mice were treated per-orally, twice daily for 14 days with Lon protease (250 μg/ml) or received PBS (Sham) and left until mice developed reduced mobility or significant weight loss.
[0243] Methylene Blue Staining
[0244] The opened intestinal segments were spread flat between sheets of filter paper and fixed overnight in 10% neutral buffered formalin. Formalin-fixed sections were transferred to 70% ethanol and stained with 0.2% methylene blue. Stained sections were rinsed in deionized water and imaged by dissecting microscope.
[0245] Histology and Immunohistochemistry
[0246] Bladders were embedded in O.C.T. compound (VWR), and 5-μm-thick fresh cryosections on positively-charged microscope slides (Superfrost/Plus; Thermo Fisher Scientific) were fixed with 4% paraformaldehyde or acetone-methanol (1:1 v/v). Swiss rolls were fixed in 4% paraformaldehyde and then embedded in paraffin and sectioned. For hemotoxylin-eosin (H&E) staining, Richard-Allan Scientific Signature Series Hematoxylin 7211 and Eosin-Y 7111 (Thermo Fisher Scientific) were used to counterstain the tissue sections. Imaging was done with AX10 (Carl Zeiss). Sections were permeabilized (0.25% Triton X-100, 5% fetal calf serum/PBS) and incubated with primary anti-c-MYC antibody (1:500, 4° C., overnight, ab39688, Abcam). This was followed by staining with Alexa Fluor® 488-labeled secondary goat anti-rabbit antibodies (1:200, 1 hour, room temperature, A-11011, Molecular Probes). Tissues were counterstained with (1:1000, 15 minutes, room temperature, Abcam) and examined using an LSM 510 confocal microscope (Carl Zeiss). The quantification of images and fluorescence was performed in ImageJ.
[0247] Quantification and Statistical Analysis
[0248] For data determined to follow a Gaussian distribution (D'Agostino & Pearson normality test), statistical differences were determined by two-tailed t-test or one-way ANOVA. For non-parametric data, Mann-Whitney U-tests were used. Paired data was analyzed by Wilcoxon matched pairs signed rank test. Kaplan Mayer plots were analysed using the log-rank Mantel-Cox test. Statistical significance was determined by GraphPad (Prism v. 7) and significance was assigned at *P<0.05, **P<0.01 and ***P<0.001.
[0249] Discussion
[0250] Microbes are essential constituents of most natural environments and potent regulators of tissue homeostasis and health in human hosts. Viruses enter host cells and reprogram the cellular machinery for their own benefit but bacteria often have a different strategy. As independent cells, they do not need invasion to survive or establish contact with host tissues, but can send messengers that trigger desired host cell effects. It is increasingly being realized that the messengers are not just the classical exotoxins or innate immune activators, with destructive effects, but molecules that reprogram basic cellular functions by targeting key functional elements, such as the transcriptional machinery.sup.32,33. This study identifies the pleiotropic transcription factor and oncogene c-MYC as a specific bacterial target. We show that uropathogenic E. coli infection depletes c-MYC from infected tissues and explain this effect by accelerated c-MYC protein degradation and attenuated MYC expression. The bacterial strategy for c-MYC inhibition is also shown to translate to cancer therapy, as the bacterial Lon protease responsible for MYC degradation attenuates cancer progression and increases survival. The bacterial Lon protease thus shows significant promise as a new approach to targeting MYC therapeutically.
[0251] The Lon protease is a conserved, ATP-dependent peptidase that maintains homeostasis in prokaryotes by processing short-lived regulatory proteins.sup.34. The protease was shown to degrade c-MYC in a time-dependent manner. Lon is a serine protease and serine-rich repeats in c-MYC were identified as cleavage sites by mass spectrometry. The c-MYC cleavage products had lost essential functional domains, such as the DNA-binding and C terminal MAX-binding sites. MYB expression was not directly affected but c-MYB was degraded in a CK1α1 dependent manner. MAX was affected, however, suggesting an unexpected level of specificity. The lower sensitivity to Lon degradation of MAX and upstream regulators of c-MYC may reflect their lower serine content. This specificity of Lon for c-MYC may be critical to limit the effects of c-MYC inhibition and avoid causing toxicity or a general metabolic collapse that would be expected to accompany a MYC shut-down.sup.35. Except for modest effects on MYC-dependent cellular functions, no evidence of major Lon toxicity was observed, supporting a loss of disease without a major gain of toxicity. This should be addressed further in extended studies.
[0252] The results further suggest that pathogenic bacteria regulate cellular MYC levels through a mechanism involving the pore forming toxin hemolysin (ref.). Ca.sup.2+ fluxes, which are activated by hemolysin, have previously been shown to activate CK1α1 and an E. coli 536 hly.sup.1/2 deletion mutant had lost the ability to activate CK1α1, indicating a clear role for hemolysin in CK1α1 activation. CK1α1 was also involved in MYB degradation, predicted to further attenuate MYC expression. Hemolysin was not required for Lon to be active, however, as the recombinant Lon protein alone inhibited MYC in human cells and treated tumors. It is therefore reasonable to assume that the effects of the two MYC inhibition mechanisms might be additive In tissues exposed to bacterial strains that express both Lon and Hly. The localized effects in tissues adjacent to the site of infection further suggest that it may be possible to achieve local MYC inhibition, at specific tissue sites.
[0253] Myc inhibition has convincingly been shown to arrest cancer progression and to restore tissue integrity in transgenic models.sup.35,36. This study suggests that the Lon protease holds promise as a broadly applicable anti-tumor agent. While the precise mechanism of action for Lon protease in vivo remains to be conclusively defined, gene expression analysis identified striking changes in treated tissues. The overexpression of MYC and cancer genes was reversed in most mice, resulting in disease attenuation in two models. Importantly, this response was not observed in healthy mice treated with Lon, suggesting that the MYC overexpression phenotype may define the therapeutic window of this molecule. Clearly, per-oral administration was effective, despite the complexity of the intestinal microbiota and tumor progression was delayed long-term in both models.
[0254] It is not clear why oncogenic mutations become fixed in the population, as cancer is a disease mainly of post reproductive age groups. There are good arguments that, at least in humans, cancer is not much of a selective force. With tragic exceptions, cancer is a disease of advanced age, and the incidence increases by a power of 4 or 5, as the reproductive value declines.sup.37,38. Stated another way, beneficial genes that act late in life, like those that may reduce the incidence of cancers, would provide little advantage. Severe, life-threatening infections, in contrast, are a strong selective force. Prior to the era of antibiotics, the mortality of acute pyelonephritis was around 15% and we are now seeing the re-emergence of these severe disease consequences. 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