Tumor FAK orchestrates immunosuppression in ovarian cancer via the CD155/TIGIT axis.

High-grade serous ovarian cancer (HGSOC) is a lethal malignancy characterized by an immunosuppressive tumor microenvironment containing few tumor infiltrating lymphocytes (TILs) and an insensitivity to checkpoint inhibitor immunotherapies. Gains in the PTK2 gene encoding focal adhesion kinase (FAK) at Chr8 q24.3 occur in ∼70% of HGSOC tumors, and elevated FAK messenger RNA (mRNA) levels are associated with poor patient survival. Herein, we show that active FAK, phosphorylated at tyrosine-576 within catalytic domain, is significantly increased in late-stage HGSOC tumors. Active FAK costained with CD155, a checkpoint receptor ligand for TIGIT (T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains), in HGSOC tumors and a selective association between FAK and TIGIT checkpoint ligands were supported by patient transcriptomic database analysis. HGSOC tumors with high FAK expression were associated with low CD3 mRNA levels. Accordingly, late-stage tumors showed elevated active FAK staining and significantly lower levels of CD3+ TILs. Using the KMF (Kras, Myc, FAK) syngeneic ovarian tumor model containing spontaneous PTK2 (FAK) gene gains, the effects of tumor intrinsic genetic or oral small molecule FAK inhibitior (FAKi; VS-4718) were evaluated in vivo. Blocking FAK activity decreased tumor burden, suppressed ascites KMF-associated CD155 levels, and increased peritoneal TILs. The combination of FAKi with blocking TIGIT antibody (1B4) maintained elevated TIL levels and reduced TIGIT+ T regulatory cell levels, prolonged host survival, increased CXCL13 levels, and led to the formation of omental tertiary lymphoid structures. Collectively, our studies support FAK and TIGIT targeting as a rationale immunotherapy combination for HGSOC.

A phase 1b study of zilovertamab in combination with paclitaxel for locally advanced/unresectable or metastatic Her2-negative breast cancer.

BACKGROUND: Zilovertamab is a humanized monoclonal antibody targeting ROR1, an onco-embryonic antigen expressed by malignant cells of a variety of solid tumors, including breast cancer. A prior phase 1 study showed that zilovertamab was well tolerated and effective in inhibiting ROR1-signaling, which leads to activation of ERK1/2, NF-κB, and NRF2 target genes. This phase 1b study evaluated the safety and tolerability of zilovertamab with paclitaxel in patients with advanced breast cancer. PATIENTS AND METHODS: Eligible patients had locally advanced, unresectable, or metastatic HER2- breast cancer with Eastern Cooperative Group performance status of 0-2 and without prior taxane therapy in the advanced setting. Study treatment included 600 mg of zilovertamab administered intravenously (IV) on Days 1 and 15 of Cycle 1 and then Day 1 of each 28-day cycle along with paclitaxel weekly at 80 mg/m2 IV. RESULTS: Study patients had received a median of 4 prior therapies (endocrine therapy + chemotherapy) for locally advanced, unresectable, or metastatic disease. No patient discontinued therapy due to toxicity ascribed to zilovertamab. Adverse events were consistent with the known safety profile of paclitaxel. Of 16 patients, 6 (38%) had a partial response, and 6/16 (38%) patients had stable disease as best tumor response. CONCLUSION: The combination of zilovertamab and paclitaxel was safe and well tolerated in heavily pre-treated advanced breast cancer patients. Further evaluation of ROR1 targeting in breast cancer patients with zilovertamab is warranted. TRIAL REGISTRATION: NCT02776917. Registered on ClinicalTrials.gov on 05/17/2016.

Suppressing neutrophil-dependent angiogenesis abrogates resistance to anti-VEGF antibody in a genetic model of colorectal cancer.

We tested cis-ApcΔ716/Smad4+/- and cis-ApcΔ716/Smad4+/-KrasG12D mice, which recapitulate key genetic abnormalities accumulating during colorectal cancer (CRC) tumorigenesis in humans, for responsiveness to anti-VEGF therapy. We found that even tumors in cis-ApcΔ716/Smad4+/-KrasG12D mice, although highly aggressive, were suppressed by anti-VEGF treatment. We tested the hypothesis that inflammation, a major risk factor and trigger for CRC, may affect responsiveness to anti-VEGF. Chemically induced colitis (CIC) in cis-ApcΔ716/Smad4+/- and cis-ApcΔ716/Smad4+/-KrasG12D mice promoted development of colon tumors that were largely resistant to anti-VEGF treatment. The myeloid growth factor G-CSF was markedly increased in the serum after induction of colitis. Antibodies blocking G-CSF, or its target Bv8/PROK2, suppressed tumor progression and myeloid cell infiltration when combined with anti-VEGF in CIC-associated CRC and in anti-VEGF-resistant CRC liver metastasis models. In a series of CRC specimens, tumor-infiltrating neutrophils strongly expressed Bv8/PROK2. CRC patients had significantly higher plasma Bv8/PROK2 levels than healthy volunteers and high plasma Bv8/PROK2 levels were inversely correlated with overall survival. Our findings establish Bv8/PROK2 as a translational target in CRC, in combination with anti-VEGF agents.

Progesterone receptor isoform ratio dictates antiprogestin/progestin effects on breast cancer growth and metastases: A role for NDRG1.

Progesterone receptors (PRs) ligands are being tested in luminal breast cancer. There are mainly two PR isoforms, PRA and PRB, and their ratio (PRA/PRB) may be predictive of antiprogestin response. Our aim was to investigate: the impact of the PR isoform ratio on metastatic behaviour, the PR isoform ratio in paired primary tumours and lymph node metastases (LNM) and, the effect of antiprogestin/progestins on metastatic growth. Using murine and human metastatic models, we demonstrated that tumours with PRB > PRA (PRB-H) have a higher proliferation index but less metastatic ability than those with PRA > PRB (PRA-H). Antiprogestins and progestins inhibited metastatic burden in PRA-H and PRB-H models, respectively. In breast cancer samples, LNM retained the same PRA/PRB ratio as their matched primary tumours. Moreover, PRA-H LNM expressed higher total PR levels than the primary tumours. The expression of NDRG1, a metastasis suppressor protein, was higher in PRB-H compared to PRA-H tumours and was inversely regulated by antiprogestins/progestins. The binding of the corepressor SMRT at the progesterone responsive elements of the NDRG1 regulatory sequences, together with PRA, impeded its expression in PRA-H cells. Antiprogestins modulate the interplay between SMRT and AIB1 recruitment in PRA-H or PRB-H contexts regulating NDRG1 expression and thus, metastasis. In conclusion, we provide a mechanistic interpretation to explain the differential role of PR isoforms in metastatic growth and highlight the therapeutic benefit of using antiprogestins in PRA-H tumours. The therapeutic effect of progestins in PRB-H tumours is suggested.

Pembrolizumab plus cetuximab in patients with recurrent or metastatic head and neck squamous cell carcinoma: an open-label, multi-arm, non-randomised, multicentre, phase 2 trial.

BACKGROUND: Pembrolizumab (PD-1 inhibitor) and cetuximab (EGFR inhibitor) are active as single agents and in combination with cytotoxic chemotherapy for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). Given each drug's single agent activity and unique mechanism of action, we aimed to evaluate the anti-tumour activity of PD-1 blockade with EGFR inhibition in recurrent or metastatic HNSCC. METHODS: This study is an open-label, non-randomised, multi-arm, phase 2 trial done at four academic centres in the USA. Participants were required to have platinum-resistant or platinum-ineligible, recurrent or metastatic HNSCC, be at least 18 years old, have an Eastern Cooperative Oncology Group performance status 0-1, have measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, and to have received no previous immunotherapy or EGFR inhibition. All participants received pembrolizumab 200 mg intravenously every 3 weeks, combined with an initial loading dose of cetuximab 400 mg/m2 intravenously followed by 250 mg/m2 intravenously weekly (21 day cycle). The primary endpoint was overall response rate defined as the proportion of participants with a partial or complete responses (per RECIST version 1.1) by 6 months in the intention-to-treat population. The safety population included all participants who received at least one dose of pembrolizumab. Herein, the final analysis of cohort 1 (no previous PD-1, PD-L1, or EGFR inhibition for recurrent or metastatic HNSCC) is reported. Three additional cohorts (two for participants with a previous response to immunotherapy followed by relapse or progression, with or without previous cetuximab exposure, and one for cutaneous HNSCC) will be reported separately once fully accrued. This study is registered with ClinicalTrials.gov, NCT03082534, and remains open as the three additional cohorts are actively accruing participants. FINDINGS: Between March 22, 2017, and July 16, 2019, 33 participants were enrolled to cohort 1. All 33 participants received at least one dose of pembrolizumab. Median follow-up duration was 7·3 months (IQR 3·9-10·9). By 6 months, the overall response rate was 45% (95% CI 28-62), with 15 of 33 participants achieving a partial response. The most common grade 3-4 treatment-related adverse event was oral mucositis (three [9%] of 33 participants), and serious treatment-related adverse events occurred in five (15%) participants. No treatment-related deaths occurred. INTERPRETATION: Pembrolizumab combined with cetuximab shows promising clinical activity for recurrent or metastatic HNSCC, and merits further investigation. FUNDING: Merck Sharp & Dohme.

Expression of an active Gαs mutant in skeletal stem cells is sufficient and necessary for fibrous dysplasia initiation and maintenance.

Fibrous dysplasia (FD) is a disease caused by postzygotic activating mutations of GNAS (R201C and R201H) that encode the α-subunit of the Gs stimulatory protein. FD is characterized by the development of areas of abnormal fibroosseous tissue in the bones, resulting in skeletal deformities, fractures, and pain. Despite the well-defined genetic alterations underlying FD, whether GNAS activation is sufficient for FD initiation and the molecular and cellular consequences of GNAS mutations remains largely unresolved, and there are no currently available targeted therapeutic options for FD. Here, we have developed a conditional tetracycline (Tet)-inducible animal model expressing the GαsR201C in the skeletal stem cell (SSC) lineage (Tet-GαsR201C/Prrx1-Cre/LSL-rtTA-IRES-GFP mice), which develops typical FD bone lesions in both embryos and adult mice in less than 2 weeks following doxycycline (Dox) administration. Conditional GαsR201C expression promoted PKA activation and proliferation of SSCs along the osteogenic lineage but halted their differentiation to mature osteoblasts. Rather, as is seen clinically, areas of woven bone admixed with fibrous tissue were formed. GαsR201C caused the concomitant expression of receptor activator of nuclear factor kappa-B ligand (Rankl) that led to marked osteoclastogenesis and bone resorption. GαsR201C expression ablation by Dox withdrawal resulted in FD-like lesion regression, supporting the rationale for Gαs-targeted drugs to attempt FD cure. This model, which develops FD-like lesions that can form rapidly and revert on cessation of mutant Gαs expression, provides an opportunity to identify the molecular mechanism underlying FD initiation and progression and accelerate the development of new treatment options.

Phase 1 dose-finding study of metformin in combination with concurrent cisplatin and radiotherapy in patients with locally advanced head and neck squamous cell cancer.

BACKGROUND: The 5-year overall survival (OS) rate remains at 50% for patients with locally advanced head and neck squamous cell carcinoma (LAHNSCC), thereby underscoring the need for improved treatments. An antidiabetic agent, metformin, was found in retrospective studies to improve survival in patients with HNSCC. Therefore, the authors conducted a phase 1 dose escalation study combining metformin with chemoradiotherapy in patients with LAHNSCC. METHODS: Nondiabetic patients with LAHNSCC were enrolled in the current study to receive escalating doses of metformin and CRT based on the modified toxicity probability interval design. Metformin cohort doses included 2000 mg, 2550 mg, and 3000 mg daily in divided doses in addition to cisplatin (at a dose of 100 mg/m2 on days 1, 22, and 43) and standard radiotherapy (70 grays). Adverse events were categorized as per the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.03). RESULTS: Twenty patients were enrolled, 2 of whom withdrew consent. The median age of the patients was 56 years and the majority were male (83%), were white (88%), had p16-positive disease (72%), and were tobacco users (61%). The median length of metformin exposure was 28.5 days. The most common grade ≥3 toxicities were nausea (11%), vomiting (11%), mucositis (6%), acute kidney injury (17%), anemia (6%), and leukopenia (11%). Dose-limiting toxicities included diarrhea and acute kidney injury. After a median follow-up of 19 months, the 2-year overall survival and progression-free survival rates were 90% and 84%, respectively. No hypoglycemia events or lactic acidosis were observed. Cisplatin administration did not appear to affect metformin pharmacokinetics. The maximum tolerated dose for metformin could not be determined given the limited number of patients who tolerated metformin during chemoradiotherapy. CONCLUSIONS: To the authors' knowledge, the current study is the first phase 1 trial combining metformin with chemoradiotherapy. Rates of overall survival and progression-free survival were encouraging in this limited patient population, and warrant further investigation in a phase 2 trial.

Age-related differences in breast cancer mortality according to race/ethnicity, insurance, and socioeconomic status.

BACKGROUND: We assessed breast cancer mortality in older versus younger women according to race/ethnicity, neighborhood socioeconomic status (nSES), and health insurance status. METHODS: The study included female breast cancer cases 18 years of age and older, diagnosed between 2005 and 2015 in the California Cancer Registry. Multivariable Cox proportional hazards modeling was used to generate hazard ratios (HR) of breast cancer specific deaths and 95% confidence intervals (CI) for older (60+ years) versus younger (< 60 years) patients separately by race/ethnicity, nSES, and health insurance status. RESULTS: Risk of dying from breast cancer was higher in older than younger patients after multivariable adjustment, which varied in magnitude by race/ethnicity (P-interaction< 0.0001). Comparing older to younger patients, higher mortality differences were shown for non-Hispanic White (HR = 1.43; 95% CI, 1.36-1.51) and Hispanic women (HR = 1.37; 95% CI, 1.26-1.50) and lower differences for non-Hispanic Blacks (HR = 1.17; 95% CI, 1.04-1.31) and Asians/Pacific Islanders (HR = 1.15; 95% CI, 1.02-1.31). HRs comparing older to younger patients varied by insurance status (P-interaction< 0.0001), with largest mortality differences observed for privately insured women (HR = 1.51; 95% CI, 1.43-1.59) and lowest in Medicaid/military/other public insurance (HR = 1.18; 95% CI, 1.10-1.26). No age differences were shown for uninsured women. HRs comparing older to younger patients were similar across nSES strata. CONCLUSION: Our results provide evidence for the continued disparity in Black-White breast cancer mortality, which is magnified in younger women. Moreover, insurance status continues to play a role in breast cancer mortality, with uninsured women having the highest risk for breast cancer death, regardless of age.

Oncogenic driver genes and the inflammatory microenvironment dictate liver tumor phenotype.

UNLABELLED: The majority of hepatocellular carcinoma develops in the background of chronic liver inflammation caused by viral hepatitis and alcoholic or nonalcoholic steatohepatitis. However, the impact of different types of chronic inflammatory microenvironments on the phenotypes of tumors generated by distinct oncogenes is largely unresolved. To address this issue, we generated murine liver tumors by constitutively active AKT-1 (AKT) and ß-catenin (CAT), followed by induction of chronic liver inflammation by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and carbon tetrachloride. Also, the impact of DDC-induced chronic liver inflammation was compared between two liver tumor models using a combination of AKT-CAT or AKT-NRAS(G12V) . Treatment with DDC and carbon tetrachloride significantly facilitated the adenoma-to-carcinoma conversion and accelerated the growth of AKT-CAT tumors. Furthermore, DDC treatment altered the morphology of AKT-CAT tumors and caused loss of lipid droplets. Transcriptome analysis of AKT-CAT tumors revealed that cellular growth and proliferation were mainly affected by chronic inflammation and caused up-regulation of Cxcl16, Galectin-3, and Nedd9, among others. Integration with transcriptome profiles from human hepatocellular carcinomas further demonstrated that AKT-CAT tumors generated in the context of chronic liver inflammation showed enrichment of poor prognosis gene sets or decrease of good prognosis gene sets. In contrast, DDC had a more subtle effect on AKT-NRAS(G12V) tumors and primarily enhanced already existent tumor characteristics as supported by transcriptome analysis. However, it also reduced lipid droplets in AKT-NRAS(G12V) tumors. CONCLUSION: Our study suggests that liver tumor phenotype is defined by a combination of driving oncogenes but also the nature of chronic liver inflammation. (Hepatology 2016;63:1888-1899).

Liver damage and senescence increases in patients developing hepatocellular carcinoma.

BACKGROUND AND AIM: Most patients with a hepatocellular carcinoma (HCC) have an underlying chronic liver inflammation, which causes a continuous damage leading to liver cirrhosis and eventually HCC. However, only a minority of cirrhotic patients develop HCC. To assess a possible differential impact of liver inflammation in patients developing HCC versus patients remaining tumor-free, we designed a longitudinal study and analysed liver tissue of the same patients (n = 33) at two points in time: once when no HCC was present and once several years later when an HCC was present. As a control group, we followed cirrhotic patients (n = 37) remaining tumor-free over a similar time frame. METHODS: We analysed cell damage and senescence of hepatocytes by measuring γ-H2AX positivity, p16INK4 and p21WAF/Cip1 expression, nuclear size, and telomere length. RESULTS: γ-H2AX positivity, p16INK4 and p21WAF/Cip1 expression, in the first liver biopsy was similar in patients developing HCC later on and cirrhotic patients remaining tumor free. In contrast, γ-H2AX positivity, p16INK4 and p21WAF/Cip1 expression, was significantly higher in the second non-tumoral liver biopsy of HCC patients than in the control patients. Consequently, the individual increase in γ-H2AX positivity, p16INK4 and p21WAF/Cip1 expression, from the first biopsy to the second biopsy was significantly higher in patients developing HCC than in patients remaining tumor free. In addition, changes in nuclear size and telomere length revealed a more pronounced cell aging in patients developing HCC than in patients remaining tumor free. CONCLUSIONS: Hepatocytes from patients developing HCC go through more pronounced cell damage and senescence in contrast to cirrhotic patients remaining tumor free.

A systems genetics approach identifies CXCL14, ITGAX, and LPCAT2 as novel aggressive prostate cancer susceptibility genes.

Although prostate cancer typically runs an indolent course, a subset of men develop aggressive, fatal forms of this disease. We hypothesize that germline variation modulates susceptibility to aggressive prostate cancer. The goal of this work is to identify susceptibility genes using the C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of neuroendocrine prostate cancer. Quantitative trait locus (QTL) mapping was performed in transgene-positive (TRAMPxNOD/ShiLtJ) F2 intercross males (n = 228), which facilitated identification of 11 loci associated with aggressive disease development. Microarray data derived from 126 (TRAMPxNOD/ShiLtJ) F2 primary tumors were used to prioritize candidate genes within QTLs, with candidate genes deemed as being high priority when possessing both high levels of expression-trait correlation and a proximal expression QTL. This process enabled the identification of 35 aggressive prostate tumorigenesis candidate genes. The role of these genes in aggressive forms of human prostate cancer was investigated using two concurrent approaches. First, logistic regression analysis in two human prostate gene expression datasets revealed that expression levels of five genes (CXCL14, ITGAX, LPCAT2, RNASEH2A, and ZNF322) were positively correlated with aggressive prostate cancer and two genes (CCL19 and HIST1H1A) were protective for aggressive prostate cancer. Higher than average levels of expression of the five genes that were positively correlated with aggressive disease were consistently associated with patient outcome in both human prostate cancer tumor gene expression datasets. Second, three of these five genes (CXCL14, ITGAX, and LPCAT2) harbored polymorphisms associated with aggressive disease development in a human GWAS cohort consisting of 1,172 prostate cancer patients. This study is the first example of using a systems genetics approach to successfully identify novel susceptibility genes for aggressive prostate cancer. Such approaches will facilitate the identification of novel germline factors driving aggressive disease susceptibility and allow for new insights into these deadly forms of prostate cancer.

mTOR inhibition prevents rapid-onset of carcinogen-induced malignancies in a novel inducible HPV-16 E6/E7 mouse model.

The rising incidence of human papillomavirus (HPV)-associated malignancies, especially for oropharyngeal cancers, has highlighted the urgent need to understand how the interplay between high-risk HPV oncogenes and carcinogenic exposure results in squamous cell carcinoma (SCC) development. Here, we describe an inducible mouse model expressing high risk HPV-16 E6/E7 oncoproteins in adults, bypassing the impact of these viral genes during development. HPV-16 E6/E7 genes were targeted to the basal squamous epithelia in transgenic mice using a doxycycline inducible cytokeratin 5 promoter (cK5-rtTA) system. After doxycycline induction, both E6 and E7 were highly expressed, resulting in rapid epidermal hyperplasia with a remarkable expansion of the proliferative cell compartment to the suprabasal layers. Surprisingly, in spite of the massive growth of epithelial cells and their stem cell progenitors, HPV-E6/E7 expression was not sufficient to trigger mTOR activation, a key oncogenic driver in HPV-associated malignancies, and malignant progression to SCC. However, these mice develop SCC rapidly after a single exposure to a skin carcinogen, DMBA, which was increased by the prolonged exposure to a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Thus, only few oncogenic hits may be sufficient to induce cancer in E6/E7 expressing cells. All HPV-E6/E7 expressing SCC lesions exhibited increased mTOR activation. Remarkably, rapamycin, an mTOR inhibitor, abolished tumor development when administered to HPV-E6/E7 mice prior to DMBA exposure. Our findings revealed that mTOR inhibition protects HPV-E6/E7 expressing tissues form SCC development upon carcinogen exposure, thus supporting the potential clinical use of mTOR inhibitors as a molecular targeted approach for prevention of HPV-associated malignancies.

SDF-1/CXCL12 induces directional cell migration and spontaneous metastasis via a CXCR4/Gαi/mTORC1 axis.

Multiple human malignancies rely on C-X-C motif chemokine receptor type 4 (CXCR4) and its ligand, SDF-1/CXCL12 (stroma cell-derived factor 1/C-X-C motif chemokine 12), to metastasize. CXCR4 inhibitors promote the mobilization of bone marrow stem cells, limiting their clinical application for metastasis prevention. We investigated the CXCR4-initiated signaling circuitry to identify new potential therapeutic targets. We used HeLa human cancer cells expressing high levels of CXCR4 endogenously. We found that CXCL12 promotes their migration in Boyden chamber assays and single cell tracking. CXCL12 activated mTOR (mechanistic target of rapamycin) potently in a pertussis-sensitive fashion. Inhibition of mTOR complex 1 (mTORC1) by rapamycin [drug concentration causing 50% inhibition (IC50) = 5 nM] and mTORC1/mTORC2 by Torin2 (IC50 = 6 nM), or by knocking down key mTORC1/2 components, Raptor and Rictor, respectively, decreased directional cell migration toward CXCL12. We developed a CXCR4-mediated spontaneous metastasis model by implanting HeLa cells in the tongue of SCID-NOD mice, in which 80% of the animals develop lymph node metastasis. It is surprising that mTORC1 disruption by Raptor knockdown was sufficient to reduce tumor growth by 60% and spontaneous metastasis by 72%, which were nearly abolished by rapamycin. In contrast, disrupting mTORC2 had no effect in tumor growth or metastasis compared with control short hairpin RNAs. These data suggest that mTORC1 may represent a suitable therapeutic target in human malignancies using CXCR4 for their metastatic spread. .

Nuclear staining of fgfr-2/stat-5 and runx-2 in mucinous breast cancer.

Mucinous carcinoma (MBC) is a rare subtype of breast cancer characterized by the production of variable amounts of mucin, with a prognosis better than that of non-mucinous carcinomas (NMBC). The aim of this project was to evaluate the expression of STAT-5, RUNX-2, and FGFR-2 in a cohort of MBC and compare it with that of NMBC using standard immunohistochemistry. STAT-5 and RUNX-2 are two transcription factors with cytoplasmic and/or nuclear localization that have been related to FGFR-2, a tyrosine kinase growth factor receptor that can interact with STAT-5 and with PR in the nuclei of breast cancer cells. Membranous, cytoplasmic, and nuclear staining were evaluated and expressed as the percentage of stained cells (0-100%) multiplied by the staining intensity (0-3), thus obtaining an index ranging from 0 to 300. Nuclear and/or cytoplasmic immunoreactivity of the three proteins were detected in a high number of NMBC. Nuclear FGFR-2 staining correlated with nuclear STAT-5 (p<0.05) and nuclear RUNX-2 (p<0.01) in both tumor types; however MBC had a significant higher expression of nuclear FGFR-2 (p<0.01) and RUNX-2 (p<0.05) than that of NMBC, and displayed positive immunoreactivity of the 3 proteins in 70.8% of the cases. These results suggest that these proteins may have a role in the progression of the mucinous phenotype, in which nuclear STAT-5 may inhibit RUNX-2 prometastatic effect.

Progestin and antiprogestin responsiveness in breast cancer is driven by the PRA/PRB ratio via AIB1 or SMRT recruitment to the CCND1 and MYC promoters.

There is emerging interest in understanding the role of progesterone receptors (PRs) in breast cancer. The aim of this study was to investigate the proliferative effect of progestins and antiprogestins depending on the relative expression of the A (PRA) and B (PRB) isoforms of PR. In mifepristone (MFP)-resistant murine carcinomas antiprogestin responsiveness was restored by re-expressing PRA using demethylating agents and histone deacetylase inhibitors. Consistently, in two human breast cancer xenograft models, one manipulated to overexpress PRA or PRB (IBH-6 cells), and the other expressing only PRA (T47D-YA) or PRB (T47D-YB), MFP selectively inhibited the growth of PRA-overexpressing tumors and stimulated IBH-6-PRB xenograft growth. Furthermore, in cells with high or equimolar PRA/PRB ratios, which are stimulated to proliferate in vitro by progestins, and are inhibited by MFP, MPA increased the interaction between PR and the coactivator AIB1, and MFP favored the interaction between PR and the corepressor SMRT. In a PRB-dominant context in which MFP stimulates and MPA inhibits cell proliferation, the opposite interactions were observed. Chromatin immunoprecipitation assays in T47D cells in the presence of MPA or MFP confirmed the interactions between PR and the coregulators at the CCND1 and MYC promoters. SMRT downregulation by siRNA abolished the inhibitory effect of MFP on MYC expression and cell proliferation. Our results indicate that antiprogestins are therapeutic tools that selectively inhibit PRA-overexpressing tumors by increasing the SMRT/AIB1 balance at the CCND1 and MYC promoters.

TMPRSS13 deficiency impairs stratum corneum formation and epidermal barrier acquisition.

Membrane-anchored serine proteases serve as important regulators of multiple developmental and homoeostatic processes in mammals. TMPRSS13 (transmembrane protease, serine 13; also known as mosaic serine protease large-form, MSPL) is a membrane-anchored serine protease with unknown biological functions. In the present study, we used mice with the Tmprss13 gene disrupted by a ß-galactosidase-neomycin fusion gene insertion to study the expression and function of the membrane-anchored serine protease. High levels of Tmprss13 expression were found in the epithelia of the oral cavity, upper digestive tract and skin. Compatible with this expression pattern, Tmprss13-deficient mice displayed abnormal skin development, leading to a compromised barrier function, as measured by the transepidermal fluid loss rate of newborn mice. The present study provides the first biological function for the transmembrane serine protease TMPRSS13.

Comparative toxicity and efficacy of engineered anthrax lethal toxin variants with broad anti-tumor activities.

We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma; mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA-activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32% to 87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5-3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers.

The TSC2/mTOR pathway drives endothelial cell transformation induced by the Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor.

The Kaposi's sarcoma-associated herpesvirus (KSHV), the infectious causative agent of Kaposi's sarcoma (KS), encodes a G protein-coupled receptor (vGPCR) implicated in the initiation of KS. Here we demonstrate that Kaposi's sarcomagenesis involves stimulation of tuberin (TSC2) phosphorylation by vGPCR, promoting the activation of mTOR through both direct and paracrine mechanisms. Pharmacologic inhibition of mTOR with rapamycin prevented vGPCR sarcomagenesis, while overactivation of this pathway was sufficient to render endothelial cells oncogenic. Moreover, mice haploinsufficient for TSC2 are predisposed to vascular sarcomas remarkably similar to KS. Collectively, these results implicate mTOR in KS initiation and suggest that the sarcomagenic potential of KSHV may be a direct consequence of the profound sensitivity of endothelial cells to vGPCR dysregulation of the TSC2/mTOR pathway.

RNF185 Control of COL3A1 Expression Limits Prostate Cancer Migration and Metastatic Potential.

RNF185 is a RING finger domain-containing ubiquitin ligase implicated in ER-associated degradation. Prostate tumor patient data analysis revealed a negative correlation between RNF185 expression and prostate cancer progression and metastasis. Likewise, several prostate cancer cell lines exhibited greater migration and invasion capabilities in culture upon RNF185 depletion. Subcutaneous inoculation of mouse prostate cancer MPC3 cells stably expressing short hairpin RNA against RNF185 into mice resulted in larger tumors and more frequent lung metastases. RNA-sequencing and Ingenuity Pathway Analysis identified wound-healing and cellular movement among the most significant pathways upregulated in RNF185-depleted lines, compared with control prostate cancer cells. Gene Set Enrichment Analyses performed in samples from patients harboring low RNF185 expression and in RNF185-depleted lines confirmed the deregulation of genes implicated in epithelial-to-mesenchymal transition. Among those, COL3A1 was identified as the primary mediator of RNF185's ability to impact migration phenotypes. Correspondingly, enhanced migration and metastasis of RNF185 knockdown (KD) prostate cancer cells were attenuated upon co-inhibition of COL3A1. Our results identify RNF185 as a gatekeeper of prostate cancer metastasis, partly via its control of COL3A1 availability. IMPLICATIONS: RNF185 is identified as an important regulator of prostate cancer migration and metastasis, in part due to its regulation of COL3A1. Both RNF185 and COL3A1 may serve as novel markers for prostate tumors.