The c-MYC-BMI1 axis is essential for SETDB1-mediated breast tumourigenesis.

Characterising the activated oncogenic signalling that leads to advanced breast cancer is of clinical importance. Here, we showed that SET domain, bifurcated 1 (SETDB1), a histone H3 lysine 9 methyltransferase, is aberrantly expressed and behaves as an oncogenic driver in breast cancer. SETDB1 enhances c-MYC and cyclin D1 expression by promoting the internal ribosome entry site (IRES)-mediated translation of MYC/CCND1 mRNA, resulting in prominent signalling of c-MYC to promote cell cycle progression, and provides a growth/self-renewal advantage to breast cancer cells. The activated c-MYC-BMI1 axis is essential for SETDB1-mediated breast tumourigenesis, because silencing of either c-MYC or BMI1 profoundly impairs the enhanced growth/colony formation conferred by SETDB1. Furthermore, c-MYC directly binds to the SETDB1 promoter region and enhances its transcription, suggesting a positive regulatory interplay between SETDB1 and c-MYC. In this study, we identified SETDB1 as a prominent oncogene and characterised the underlying mechanism whereby SETDB1 drives breast cancer, providing a therapeutic rationale for targeting SETDB1-BMI1 signalling in breast cancer. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Functional screen identifies kinases driving prostate cancer visceral and bone metastasis.

Mutationally activated kinases play an important role in the progression and metastasis of many cancers. Despite numerous oncogenic alterations implicated in metastatic prostate cancer, mutations of kinases are rare. Several lines of evidence suggest that nonmutated kinases and their pathways are involved in prostate cancer progression, but few kinases have been mechanistically linked to metastasis. Using a mass spectrometry-based phosphoproteomics dataset in concert with gene expression analysis, we selected over 100 kinases potentially implicated in human metastatic prostate cancer for functional evaluation. A primary in vivo screen based on overexpression of candidate kinases in murine prostate cells identified 20 wild-type kinases that promote metastasis. We queried these 20 kinases in a secondary in vivo screen using human prostate cells. Strikingly, all three RAF family members, MERTK, and NTRK2 drove the formation of bone and visceral metastasis confirmed by positron-emission tomography combined with computed tomography imaging and histology. Immunohistochemistry of tissue microarrays indicated that these kinases are highly expressed in human metastatic castration-resistant prostate cancer tissues. Our functional studies reveal the strong capability of select wild-type protein kinases to drive critical steps of the metastatic cascade, and implicate these kinases in possible therapeutic intervention.

Activation of Notch1 synergizes with multiple pathways in promoting castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-specific mortality. Defining new mechanisms that can predict recurrence and drive lethal CRPC is critical. Here, we demonstrate that localized high-risk prostate cancer and metastatic CRPC, but not benign prostate tissues or low/intermediate-risk prostate cancer, express high levels of nuclear Notch homolog 1, translocation-associated (Notch1) receptor intracellular domain. Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early disease to promote the development of prostate adenocarcinoma. These tumors display features of epithelial-to-mesenchymal transition, a cellular state associated with increased tumor aggressiveness. Consistent with its activation in clinical CRPC, tumors driven by Notch1 intracellular domain in combination with multiple pathways altered in prostate cancer are metastatic and resistant to androgen deprivation. Our study provides functional evidence that the Notch1 signaling axis synergizes with alternative pathways in promoting metastatic CRPC and may represent a new therapeutic target for advanced prostate cancer.

Effects of calorie restriction and IGF-1 receptor blockade on the progression of 22Rv1 prostate cancer xenografts.

Calorie restriction (CR) inhibits prostate cancer progression, partially through modulation of the IGF axis. IGF-1 receptor (IGF-1R) blockade reduces prostate cancer xenograft growth. We hypothesized that combining calorie restriction with IGF-1R blockade would have an additive effect on prostate cancer growth. Severe combined immunodeficient mice were subcutaneously injected with 22Rv1 cells and randomized to: (1) Ad libitum feeding/intraperitoneal saline (Ad-lib); (2) Ad-lib/20 mg/kg twice weekly, intraperitoneal ganitumab [anti-IGF-1R antibody (Ad-lib/Ab)]; (3) 40% calorie restriction/intraperitoneal saline (CR); (4) CR/ intraperitoneal ganitumab, (CR/Ab). CR and ganitumab treatment were initiated one week after tumor injection. Euthanasia occurred 19 days post treatment. Results showed that CR alone decreased final tumor weight, plasma insulin and IGF-1 levels, and increased apoptosis. Ganitumab therapy alone reduced tumor growth but had no effect on final tumor weight. The combination therapy (CR/Ab) further decreased final tumor weight and proliferation, increased apoptosis in comparison to the Ad-lib group, and lowered plasma insulin levels relative to the Ad-lib and Ad-lib/Ab groups. Tumor AKT activation directly correlated with plasma IGF-1 levels. In conclusion, whereas ganitumab therapy modestly affected 22Rv1 tumor growth, combining IGF-1R blockade with calorie restriction resulted in a significant decrease in final tumor weight and improved metabolic profile.

Impact of pathological tumour characteristics in patients with sarcomatoid renal cell carcinoma.

UNLABELLED: Study Type - Prognosis (case series) Level of Evidence 4 What's known on the subject? and What does the study add? Sarcomatoid renal cell carcinoma can occur in the setting of all histological subtypes of kidney cancer. These tumours are very aggressive and many patients present with disseminated disease. Long-term survival is poor and the durable responses to systemic therapy are infrequent. Our large cohort analyses the influence of pathological tumour characteristics in determining prognosis for patients with sarcomatoid renal cell carcinoma undergoing surgical resection. This series helps define the prognostic influence of histological subtype, type of sarcomatoid morphology, the percentage necrosis and sarcomatoid features, and the presence of microvascular invasion. OBJECTIVES: To examine the influence of pathological tumour characteristics on survival to aid prognostication and clinical trial design. Patients with sarcomatoid renal cell carcinoma (sRCC) are known to have poor prognosis and response to systemic therapy. PATIENTS AND METHODS: A single-centre database was reviewed to identify all patients with sRCC. Clinical variables and pathological information, including histology, necrosis, percentage of sarcomatoid features (PSF) and microvascular invasion (MVI), were recorded and correlated to outcome. RESULTS: Analyses of 104 patients with sRCC found that the median (range) size of tumours was 9.5 cm (2.5-30), 65% of patients had areas of clear cell histology, and 69.2% had metastatic disease at presentation. The PSF did not influence tumour size, stage, necrosis, MVI, nodes or metastasis. A total of 85 patients (81.7%) died during the follow-up period with a median (95% confidence interval [CI]) survival of 5.9 months (4.7-8.9). In the overall cohort, Eastern Cooperative Group performance status (ECOGPS), tumour size and metastatic disease were independent predictors of poor survival. MVI, PSF and percentage necrosis were strongly associated with outcome but were not independent predictors of outcome. A multivariate risk model was established that incorporated six covariates (tumour size, MVI, ECOGPS, PSF, necrosis, and metastatic disease) to produce a predictive tool. CONCLUSIONS: Both patients with localized and metastatic sRCC have very poor survival outcomes. Pathological features MVI, PSF and necrosis are important predictors of survival and could be used in a prognostic model while grade and histology do not influence prognosis. A prognostic model, if validated, could aid in patient counselling and/or clinical trial design.

The chromophobe tumor grading system is the preferred grading scheme for chromophobe renal cell carcinoma.

PURPOSE: The prognostic usefulness of the Fuhrman nuclear grading system has been questioned for chromophobe renal cell carcinoma due to its frequent nuclear and nucleolar pleomorphism. Chromophobe tumor grade, a novel 3-tier tumor grading system based on geographic nuclear crowding and anaplasia, was recently reported to be superior to the Fuhrman system. We compared the 2 scoring systems in a large sporadic chromophobe renal cell carcinoma cohort to determine which grading scheme provides the most predictive assessment of clinical risk. MATERIALS AND METHODS: We identified a total of 84 cases of sporadic chromophobe renal cell carcinoma in 82 patients from a total of 2,634 cases (3.2%) spanning 1989 to 2010. A subset of 11 tumors had secondary areas of sarcomatoid transformation. All cases were reviewed for Fuhrman nuclear grade and chromophobe tumor grade according to published parameters by an expert genitourinary pathologist blinded to clinicopathological information. RESULTS: The distribution of Fuhrman nuclear grades 1 to 4 was 0%, 52.4%, 32.9% and 14.7% of cases, and the distribution of chromophobe tumor grades 1 to 3 was 48.8%, 36.5% and 14.7%, respectively. Metastasis developed in 20 patients (24.4%). Survival analysis revealed statistically significant differences in recurrence-free survival when adjusted for chromophobe tumor grade and Fuhrman nuclear grade. Chromophobe tumor grade showed a slightly higher AUC for recurrence-free survival and overall survival than the Fuhrman nuclear grading system. Neither chromophobe tumor grade nor Fuhrman nuclear grade was retained as an independent predictor of outcome in multivariate modeling when patients with sarcomatoid lesions were excluded. CONCLUSIONS: Chromophobe tumor grade effectively stratifies patients with chromophobe renal cell carcinoma across all grading levels. Since it does not rely on nuclear features, it avoids the hazard of overestimating the malignant potential of chromophobe renal cell carcinoma. Overall chromophobe tumor grade has higher predictive accuracy than the Fuhrman nuclear grading system.

Stromal immune infiltration in HIV-related diffuse large B-cell lymphoma is associated with HIV disease history and patient survival.

OBJECTIVE: Understanding tumor microenvironment and its impact on prognosis of HIV-related lymphomas may provide insight into novel therapeutic strategies. DESIGN: We characterized the relationship between infiltrating immune cells with tumor characteristics, HIV disease history and survival in 80 patients with HIV-related diffuse large B-cell lymphoma (DLBCL) diagnosed in the era of combined antiretroviral therapy (1996-2007) at Kaiser Permanente California. Eighty patients with HIV-unrelated DLBCL were included for comparison. METHODS: Data on patients' clinical history were obtained from Kaiser Permanente's electronic health records. The density of stromal CD4, CD8 and FOXP3 T cells and CD68 macrophages, as well as tumor molecular characteristics were examined using immunohistochemistry. The associations between stromal immune infiltration and patient's clinical history or tumor characteristics were examined using Kruskal-Wallis tests or Pearson's correlation coefficient. The effect of stromal immune infiltration on 2-year mortality was evaluated in multivariable logistic regression. RESULTS: Compared with HIV-unrelated DLBCL, patients with HIV-related DLBCL had significantly reduced stromal CD4 and FOXP3 T cells, but increased density of macrophages. Increased density of stromal macrophages was correlated with lower circulating CD4 cell count at DLBCL diagnosis. Tumor molecular characteristics, including BCL6, p53 and cMYC expression, but not Epstein-Barr virus infection status, were significantly correlated with stromal immune infiltration, particularly FOXP3 T cells. A higher density of infiltrating CD8 T cell was significantly associated with reduced mortality in patients with HIV-related DLBCL (odds ratio = 0.30 [0.09-0.97] for ≥25 vs. <10%). CONCLUSION: These data provide evidence for the prognostic significance of cytotoxic T cells in determining outcomes of HIV-related lymphoma.

A comparative study of molecular characteristics of diffuse large B-cell lymphoma from patients with and without human immunodeficiency virus infection.

PURPOSE: HIV-related diffuse large B-cell lymphoma (DLBCL) may be biologically different from DLBCL in the general population. We compared, by HIV status, the expression and prognostic significance of selected oncogenic markers in DLBCL diagnosed at Kaiser Permanente in California, between 1996 and 2007. EXPERIMENTAL DESIGN: Eighty HIV-infected DLBCL patients were 1:1 matched to 80 HIV-uninfected DLBCL patients by age, gender, and race. Twenty-three markers in the following categories were examined using IHC: (i) cell-cycle regulators, (ii) B-cell activators, (iii) antiapoptotic proteins, and (iv) others, such as IgM. Tumor marker expression was compared across HIV infection status by Fisher exact test. For markers differentially expressed in HIV-related DLBCL, logistic regression was used to evaluate the association between tumor marker expression and 2-year overall mortality, adjusting for International Prognostic Index, cell-of-origin phenotype, and DLBCL morphologic variants. RESULTS: Expression of cMYC (% positive in HIV-related and -unrelated DLBCL: 64% vs. 32%), BCL6 (45% vs. 10%), PKC-ß2 (61% vs. 4%), MUM1 (59% vs. 14%), and CD44 (87% vs. 56%) was significantly elevated in HIV-related DLBCLs, whereas expression of p27 (39% vs. 75%) was significantly reduced. Of these, cMYC expression was independently associated with increased 2-year mortality in HIV-infected patients [relative risk = 3.09 (0.90-10.55)] in multivariable logistic regression. CONCLUSIONS: These results suggest that HIV-related DLBCL pathogenesis more frequently involves cMYC and BCL6 among other factors. In particular, cMYC-mediated pathogenesis may partly explain the more aggressive clinical course of DLBCL in HIV-infected patients.

Polyphenols in brewed green tea inhibit prostate tumor xenograft growth by localizing to the tumor and decreasing oxidative stress and angiogenesis.

It has been demonstrated in various animal models that the oral administration of green tea (GT) extracts in drinking water can inhibit tumor growth, but the effects of brewed GT on factors promoting tumor growth, including oxidant damage of DNA and protein, angiogenesis and DNA methylation, have not been tested in an animal model. To explore these potential mechanisms, brewed GT was administered instead of drinking water to male severe combined immunodeficiency (SCID) mice with androgen-dependent human LAPC4 prostate cancer cell subcutaneous xenografts. Tumor volume was decreased significantly in mice consuming GT, and tumor size was significantly correlated with GT polyphenol (GTP) content in tumor tissue. There was a significant reduction in hypoxia-inducible factor 1-alpha and vascular endothelial growth factor protein expression. GT consumption significantly reduced oxidative DNA and protein damage in tumor tissue as determined by 8-hydroxydeoxyguanosine/deoxyguanosine ratio and protein carbonyl assay, respectively. Methylation is known to inhibit antioxidative enzymes such as glutathione S-transferase pi to permit reactive oxygen species promotion of tumor growth. GT inhibited tumor 5-cytosine DNA methyltransferase 1 mRNA and protein expression significantly, which may contribute to the inhibition of tumor growth by reactivation of antioxidative enzymes. This study advances our understanding of tumor growth inhibition by brewed GT in an animal model by demonstrating tissue localization of GTPs in correlation with inhibition of tumor growth. Our results suggest that the inhibition of tumor growth is due to GTP-mediated inhibition of oxidative stress and angiogenesis in the LAPC4 xenograft prostate tumor in SCID mice.

Effect of a low-fat diet combined with IGF-1 receptor blockade on 22Rv1 prostate cancer xenografts.

In preclinical models, both dietary fat reduction and insulin-like growth factor I receptor (IGF-1R) blockade individually inhibit prostate cancer xenograft growth. We hypothesized that a low-fat diet combined with IGF-1R blockade would cause additive inhibition of prostate cancer growth and offset possible untoward metabolic effects of IGF-1R blockade antibody therapy. Fifty severe combined immunodeficient mice were injected with 22Rv1 cells subcutaneously. Ten days postinjection, the animals were randomized to four groups: (i) high-fat diet + saline (HF); (ii) high-fat diet + IGF-1R blocking antibody, ganitumab (HF/Ab); (iii) low-fat diet + saline (LF); and (iv) low-fat diet + ganitumab (LF/Ab). After 19 days of treatment, the animals were euthanized, serum was collected, and tumors were weighed. Tumor Ki67, Akt and extracellular signal-regulated kinase (ERK) activation, serum insulin, IGF-I and TNF-α were measured. In vitro, ganitumab treatment inhibited growth and induced apoptosis in several prostate cancer cell lines. In vivo, tumor weights and volumes were unaffected by the different treatments. The LF/Ab therapy significantly reduced proliferation (Ki67) and ERK activation in tumors. The HF/Ab group had significantly higher serum insulin levels than the HF group. However, LF/Ab combination significantly reduced serum insulin back to normal levels as well as normalizing serum TNF-α level. Whereas the combination of low-fat diet and IGF-1R blockade did not have additive inhibitory effects on tumor weight, it led to reduced tumor cell proliferation and a reduction in serum insulin and TNF-α levels.

Phase II prospective randomized trial of a low-fat diet with fish oil supplementation in men undergoing radical prostatectomy.

Preclinical studies suggest lowering dietary fat and decreasing the ratio of omega-6 to omega-3 polyunsaturated fatty acids decreases the risk of prostate cancer development and progression. We conducted a phase II randomized trial to test the effect of decreasing dietary fat combined with decreasing the dietary omega-6:omega-3 ratio on biomarkers related to prostate cancer development and progression. Patients undergoing radical prostatectomy were randomly assigned to receive a low-fat diet with 5 grams of fish oil daily (dietary omega-6:omega-3 ratio of 2:1) or a control Western diet (omega-6:omega-3 ratio of 15:1) for four to six weeks prior to surgery. The primary endpoint was change in serum insulin-like growth factor I (IGF-1) between arms. Secondary endpoints were serum IGFBP-1, prostate prostaglandin E2 levels, omega-6:omega-3 fatty acid ratios, COX-2, and markers of proliferation and apoptosis. Fifty-five patients were randomized and 48 completed the trial. There was no treatment difference in the primary outcome. Positive secondary outcomes in the low-fat fish oil versus Western group were reduced benign and malignant prostate tissue omega-6:omega-3 ratios, reduced proliferation (Ki-67 index), and reduced proliferation in an ex vivo bioassay when patient sera was applied to prostate cancer cells in vitro. In summary, four to six weeks of a low-fat diet and fish oil capsules to achieve an omega-6:omega-3 fatty acid ratio of 2:1 had no effect on serum IGF-1 levels, though in secondary analyses, the intervention resulted in decreased prostate cancer proliferation and decreased prostate tissue omega-6:omega-3 ratios. These results support further studies evaluating reduction of dietary fat with fish oil supplementation on modulating prostate cancer biology.

Cucurbitacin B, a novel in vivo potentiator of gemcitabine with low toxicity in the treatment of pancreatic cancer.

BACKGROUND AND PURPOSE: Pancreatic cancer is a highly aggressive malignancy, and improvement in systemic therapy is necessary to treat this frequently encountered metastatic disease. The current targeted agents used in combination with gemcitabine improved objective response rates, but with little or no improvements in survival and also increased toxicities in pancreatic cancer patients. Recently, we showed that the triterpenoid cucurbitacin B inhibited tumour growth in pancreatic cancer cells by inhibition of the JAK/STAT pathway, and synergistically increased antiproliferative effects of gemcitabine in vitro. EXPERIMENTAL APPROACH: The anti-tumour effects and toxicities of cucurbitacin B in combination with gemcitabine were tested against human pancreatic cancer cells in a murine xenograft model. KEY RESULTS: Combined therapy with cucurbitacin B and gemcitabine at relatively low doses (0.5 mg x kg(-1) and 25 mg x kg(-1) respectively) resulted in highly significant tumour growth inhibition of pancreatic cancer xenografts (up to 79%). Remarkably, this therapy was well tolerated by the animals, as shown by histology of visceral organs, analysis of serum chemistry, full blood counts and bone marrow colony numbers. Western blot analysis of the tumour samples of mice who received both cucurbitacin B and gemcitabine, revealed stronger inhibition of Bcl-XL, Bcl-2 and c-myc, and higher activation of the caspase cascades, than mice treated with either agent alone. CONCLUSIONS AND IMPLICATIONS: Combination of cucurbitacin B and gemcitabine had profound anti-proliferative effects in vivo against xenografts of human pancreatic cancer cells, without any significant signs of toxicity. This promising combination should be examined in therapeutic trials of pancreatic cancer.