Venkatesh Kolluru

Oral administration of withaferin A inhibits carcinogenesis of prostate in TRAMP model

We previously reported that withaferin A (WA), a natural compound, deters prostate cancer by inhibiting AKT while inducing apoptosis. In the current study, we examined its chemopreventive efficacy against carcinogenesis in the prostate using the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Two distinct sets of experiments were conducted. To determine whether WA delays tumor progression, it was given before cancer onset, at week 6, and until week 44. To determine its effect after the onset of prostate cancer, it was given from weeks 12 to 35. In both strategies, oral administration of WA effectively suppressed tumor burden when compared to vehicle-treated animals. No toxicity was seen in treated animals at gross pathological examination. Western blot analysis and immunohistochemistry of tumor sections revealed that in TRAMP controls, AKT and pAKT were highly expressed while nuclear FOXO3a and Par-4 were downregulated. On the contrary, treated mice showed inhibition of AKT signaling and activation of FOX03a-Par-4-induced cell death. They also displayed inhibition of mesenchymal markers such as β-catenin, vimentin, and snail as well as upregulation of E-cadherin. Because expressions of the angiogenic markers factor VIII and retic were downregulated, an anti-angiogenic role of WA is suggested. Overall, our results suggest that WA could be a promising anti-cancer agent that effectively inhibits carcinogenesis of the prostate.

miR-301a expression: A prognostic marker for prostate cancer.

PURPOSE The diagnosis and treatment of prostate cancer (CaP) continues to be challenging, as prostate-specific antigen (PSA) appears to be overly sensitive and biopsy is the only reliable method for confirmation. Hence, the goal of the study is to identify a biomarker that could distinguish malignant cancer from benign prostatic hyperplasia (BPH) during the early diagnosis of the disease. MATERIALS AND METHODS A total of 75 formalin fixed paraffin embedded (FFPE) with matching controls, 4 paired metastatic tumors, 6 fresh tumor tissues and BPH (13 cases) with their clinical diagnosis were selected for this study. Prostate cancer cell lines and normal prostate epithelial cell lines were obtained from ATCC and subjected to phenotypic analysis. RESULTS We observed significant differential expression of miR-301a in CaP samples in comparison to BPH and adjacent benign samples. The overexpression of miR-301a activates the invasion/migration of CaP cells. In contrast, silencing miR-301a expression inhibited the colony-forming ability, adhesion, invasion and migration of CaP cells. Similarly, the overexpression of miR-301a increased cell motility in normal RWPE-1 prostate epithelial cells. Our results suggest that miR-301a is differentially expressed between BPH and CaP specimens and that the expression of miR-301a correlates with biochemical recurrence and/or metastasis in CaP patients. CONCLUSIONS The expression of miR-301a could be a potential marker for metastasis in CaP patients. Detecting miR-301a expression during diagnosis will avoid wait and watch timelines, thus preventing morbidity.

Targeting aberrant expression of Notch-1 in ALDH+ cancer stem cells in breast cancer†

We have previously reported that high aldehyde dehydrogenase (ALDH) enzyme activity in breast cancer cells results in breast cancer stem cell (BCSC) properties by upregualting Notch‐1 and epithelial mesenchymal markers. This results in chemoresistance in breast cancer. Here, we examined the functional and clinical significance of ALDH expression by measuring the ALDH levels in breast cancer tissues by immunohistochemistry. There was a significantly higher ALDH expression in higher grade breast cancer tumor tissues (Grade‐ II and III) versus normal breast tissues. Injection of BCSC (ALDH+ and CD44+/CD22−) cells resulted in aggressive tumor growth in athymic mice versus ALDH− cells. The ALDH+ and CD44+/CD22− tumors grow rapidly and are larger than ALDH− tumors which were slow growing and smaller. Molecularly, ALDH+ tumors expressed higher expression of Notch‐1 and EMT markers than ALDH− tumors. Oral administration of the naturally occurring Psoralidin (Pso, 25 mg/kg of body weight) significantly inhibited the growth in ALDH+ and ALDH− tumors as well. Psoralidin inhibited Notch‐1 mediated EMT activation in ALDH+ and ALDH− tumors‐this confirms our in vitro findings. Our results suggest that Notch‐1 could be an attractive target and inhibition of Notch‐1 by Psoralidin may prevent pathogenesis of breast cancer as well as metastasis.

miR-301a expression: Diagnostic and prognostic marker for prostate cancer

BACKGROUND Prostate-specific antigen screening for prostate cancer (CaP) remains controversial. This study establishes the role of microRNA 301a (miR-301a) as a supplemental biomarker that can distinguish between patients with benign prostate hyperplasia and clinically significant CaP. We evaluate the ability of miR-301a to predict the adverse pathology of CaP. METHODS In the first cohort, serum and prostate tumor samples were obtained from thirteen patients with Benign prostate hyperplasia (BPH), twelve patients with Gleason 6, and sixteen patients with Gleason 7 prostate adenocarcinoma. In the second cohort, 40 prostatectomy cases were selected (BPH:12, Gleason 6:12 and Gleason 7:16). MiRNA was extracted from serum and tumor samples. Quantitative reverse transcription-polymerase chain reaction was performed for detection of miR-301a. To understand the molecular role of miR-301a, we performed cell viability, Western blots, promoter analysis, overexpression, and silencing studies in BPH and DU-145 cell lines. RESULTS MiR-301a demonstrated a significantly higher expression in both serum and tumor tissue in patients with CaP when compared to patients with BPH (P = 0.011 and 0.013 for serum and tissue expression, respectively). Expression of miR-301a in prostatectomy specimens correlated with increased Gleason score. We demonstrated that miR-301a inhibited the pro-apoptotic function of RUNX3, and activated ROCK1-mediated pro-survival signal in CaP. Silencing miR-301a initiated the pro-apoptotic function of RUNX3 by inhibiting ROCK1 expression in CaP cells. CONCLUSIONS Expression of miR-301a could be a valuable adjunct tool for stratifying patients with elevated prostate-specific antigen, as well as those diagnosed with CaP. Including the miR-301a as an additional variable in MSKCC post-prostatectomy nomogram improved its ability in facilitating clinical decision-making.

A natural molecule, urolithin A, downregulates androgen receptor activation and suppresses growth of prostate cancer

Androgen ablation therapy is the primary therapeutic option for locally advanced and metastatic castration‐resistant prostate cancer (CRPC). We investigated therapeutic effect of a dietary metabolite Urolithin A (UroA) and dissected the molecular mechanism in CRPC cells. Treatment with UroA inhibited cell proliferation in both androgen receptor‐positive (AR+) (C4‐2B) and androgen receptor‐negative (AR−) (PC‐3) cells however, AR+ CaP cells were more sensitive to UroA treatment as compared with AR− CaP cells. Inhibition of the AR signaling was responsible for the UroA effect on AR+ CaP cells. Ectopic expression of AR in PC‐3 cells sensitized them to UroA treatment as compared to the vector‐expresseing PC‐3 cells, which suggests that AR could be a target of UroA. Similarly, in enzalutamide‐resistant C4‐2B cells, a downregulation of AR expression also suppressed cell proliferation which was observed with the UroA treatment. Oral administration of UroA significantly suppressed the growth of C4‐2B xenografts (P = 0.05) compared with PC‐3 xenografts (P = 0.069) without causing toxicity to animals. Immunohistochemistry analysis confirmed in vitro findings such as downregulation of AR/pAKT signaling in UroA‐treated C4‐2B tumors, which suggests that UroA may be a potent chemo‐preventive and therapeutic agent for CRPC.

The chemopreventive effect of withaferin A on spontaneous and inflammation-associated colon carcinogenesis models

Chemopreventive effects and associated mechanisms of withaferin A (WA) against intestinal and colon carcinogenesis remain unknown. We investigated the chemopreventive effect of WA on transgenic adenomatous polyposis coli (APCMin/+) mouse and chemically induced azoxymethane/dextran sodium sulfate (AOM/DSS) models of intestinal and colon carcinogenesis. Oral WA administration (4 and 3 mg/kg) inhibited tumor initiation and progression of intestinal polyps formation in APCMin/+ mice and colon carcinogenesis in the AOM/DSS mouse model. WA-administered mice showed a significant reduction in both number [duodenum, 33% (P > 0.05); jejunum, 32% (P < 0.025); ileum, 43% ( P < 0.001); and colon 59% (P < 0.01] and size of polyps in APCMin/+ mice compared with the respective controls. Similarly, tumor multiplicity was significantly reduced (P < 0.05) in the colon of WA-administered AOM/DSS mice. Pathological analysis showed reduced adenomas and tissue inflammation in WA-administered mouse models. Molecular studies suggested that WA inhibited the expression of inflammatory (interluekin-6, tumor necrosis factor-alpha and cyclooxygenase-2), pro-survival (pAKT, Notch1 and NF-κB) markers in APCMin/+ and AOM/DSS models. The results suggest that WA is a potent agent for preventing colon carcinogenesis and further investigation is required to show clinical utility of the agent.

Abstract 2903: 1-Methoxyphaseollidin: Novel gamma secretase inhibitor targeting notch-1 signaling in breast cancer stem cells

We recently showed that two different ALDH+ and CD44+/CD24-/low breast cancer stem cells (BSCSs) exhibited stem cell characteristics that include self-renewal, extensive proliferation, the ability to form non-adherent spherical clusters, chemotherapy resistance and high Notch1 expression. We have identified a compound compound: 6-(3-methylbut-2-enyl) coumestrol (Pso) and treatment with Pso resulted in growth inhibition and an EMT phenotype in both BCSCs and BC cells. Oral Pso administration at physiologically achievable doses (25 mg/kg/BW) suppressed the growth of BCSCs and BC xenografts without toxicity. In the current studies, we identified several novel Pso-derived analogs that may be more potent than the parent compound. One such compound, 1-methoxyphaseollidin (1MP), obtained via three main functional group changes: (i) translocation of the isoprenyl moiety from the phenyl ring fused to the pyran ring (as in Pso) to the phenyl ring adjacent to the furan ring, (ii) removal of the carbonyl group from the pyran ring, and (iii) introduction of a methoxy group at the 1-position, inhibited Notch1 activity and growth of both BSCS and BC cells at nM concentration (IC50: 300nM), which is 100 times more potently than Pso in cell culture models. Molecular studies suggest that 1MP inhibits Notch signaling pathways (Hes1, Hey1 and Presenilin) in both BCSC and BC cells. Further, downregulation of AKT signaling (pAKT (S473), p65 and BCl-2 were seen in 1MP treated cells. Docking studies suggest that 1MP binds outside of the catalytic unit of γ-secretase and induces a conformational change, resulting in Notch1 inhibition in both BCSCs and BC cells. More importantly, administration of 1MP significantly inhibited the growth of BCSC and BC tumors without causing gastrointestinal toxicity in tumor-bearing mice. HE 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2903. doi:10.1158/1538-7445.AM2017-2903

Abstract 5445: Inhibition of tumor suppressor function of RUNX3 by miR-301a facilitates the progression of castration resistant prostate cancer

Treatment of prostate cancer is still clinically challenging due to lack of reliable markers for diagnosis and prognosis. Serum prostate specific antigen (PSA) is of limited clinical utility due to poor sensitivity and specificity. A large number of false positives (50%) with traditional PSA testing lead many patients to undergoing unnecessary prostate biopsy and exposure to unnecessary risk of complications. So the goal of this study is to identify a reliable, non-invasive biomarker that can distinguish patients with benign, indolent and aggressive prostate cancer in various clinical settings. MicroRNAs (miRNAs) are small noncoding (18 to 28 nucleotides) RNA molecules that are present in all human cells, and their expression patterns are correlated with many cancer types, including CaP. In our results, we found a significant differential expression (p=0.013) of miR-301a in both tumor tissues (Gleason-6 and -7) and serum samples in comparison to benign prostatic hyperplasia (BPH) or adjacent benign samples. We observed a negative correlation between miR-301a and RUNX3 expression in human CaP samples suggesting tumor suppressive role RUNX3 might be compromised in CaP. To determine MiR-301a regulation on RUNX3, we evaluated miR-301a and RUNX3 protein in panel of prostate cancer cell lines, normal prostate epithelial cells and Benign Prostate Hyperplasia (BPH). Similar results such as inverse correlation between miR-301a and RUNX were found in cell culture models. Also, over expression of miR-301a down regulates RUNX3 activation in prostate cancer cell lines and silencing miR-301a expression reverts RUNX3 activation in BPH cells. While evaluating the reporter activity of RUNX3 either by co-transfecting with mimic or inhibitor of miR-301a in BPH and prostate cancer cells: over expression of miR-301a down regulated RUNX3 reporter activity which corresponded with RUNX3 protein expression. Similarly, silencing miR-301a reverted RUNX3 promoter activity and protein expression suggesting RUNX3 is a direct target of miR-301a in CaP cells. Silencing miR-301a reverted the pro-apoptotic function of RUNX3, results in reduced colony formation, adhesion, invasion and migration of CaP cells. Investigating the mechanistic link miR-301a and RUNX3 may explore whether it could facilitate clinical decision making such as the decision to proceed with early surgery rather than active surveillance for intermediate risk prostate cancer patients deemed to be at high risk of progression. Citation Format: Venkatesh Kolluru, Balaji Chandrasekhar, Collin McKenzie, Houda Alatassi, Murali Ankem, Chendil Damodaran. Inhibition of tumor suppressor function of RUNX3 by miR-301a facilitates the progression of castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5445. doi:10.1158/1538-7445.AM2017-5445

Inhibition of autophagy prevents cadmium-induced prostate carcinogenesis

Background:Cadmium, an established carcinogen, is a risk factor for prostate cancer. Induction of autophagy is a prerequisite for cadmium-induced transformation and metastasis. The ability of Psoralidin (Pso), a non-toxic, orally bioavailable compound to inhibit cadmium-induced autophagy to prevent prostate cancer was investigated.Methods:Psoralidin was studied using cadmium-transformed prostate epithelial cells (CTPE), which exhibit high proliferative, invasive and colony forming abilities. Gene and protein expression were evaluated by qPCR, western blot, immunohistochemistry and immunofluorescence. Xenograft models were used to study the chemopreventive effects in vivo.Results:Cadmium-transformed prostate epithelial cells were treated with Pso resulting in growth inhibition, without causing toxicity to normal prostate epithelial cells (RWPE-1). Psoralidin-treatment of CTPE cells inhibited the expression of Placenta Specific 8, a lysosomal protein essential for autophagosome and autolysosome fusion, which resulted in growth inhibition. Additionally, Pso treatment caused decreased expression of pro-survival signalling proteins, NFκB and Bcl2, and increased expression of apoptotic genes. In vivo, Pso effectively suppressed CTPE xenografts growth, without any observable toxicity. Tumours from Pso-treated animals showed decreased autophagic morphology, mesenchymal markers expression and increased epithelial protein expression.Conclusions:These results confirm that inhibition of autophagy by Pso plays an important role in the chemoprevention of cadmium-induced prostate carcinogenesis.