Balaji Chandrasekaran

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 3930: Chemoprevention of metastatic colon cancer by a novel small molecule

Introduction: Colon cancer is a one of the leading cause of death in both men and women. Metastatic colon cancer is responsible for mortality due to resistance to conventional therapies. Our group has previously reported that activation of AKT and Notch1 plays an important role in metastasis of colon cancer cells. As Notch1 and AKT regulates cell proliferation and are upstream of epithelial to mesenchymal transition (EMT) cascade, it seems interesting to explore their inhibition for pharmacological intervention. In the present study we identified a sesquiterpene molecule, Verrucarin J (VJ), that inhibited Notch1 expression and downregulated AKT mediated EMT transition in colon cancer cells. Methods: The anticancer effect of VJ was assessed on both colon cancer cells and stably AKT overexpressing colon cells by cell proliferation, apoptosis and Western blot analysis. For xenograft studies, pCMV/HCT-116 or AKT/HCT-116 cells (1.5 × 106) in a 50-μl final volume of phosphate-buffered saline (PBS) were injected subcutaneously into separate flanks of the mice. Also effects of VJ were determined in mouse model of colorectal cancer i.e. APC min+/ _ mice. Statistical analysis was done by unpaired Student9s t-test and one way ANOVA,*p≤0.05, **p≤0.01, ***p≤0.001. Results: Molecular analysis of VJ treatment on colon cancer cells revealed that it inhibited colon cancer growth by down regulating AKT and Notch1 signaling. Western blot analysis revealed that VJ inhibits AKT/NF-κβ/ Bcl2- signaling axis in colon cancer cells. VJ also Inhibited migration and invasion of colon cancer cells that corresponds with downregulation of mesenchymal marker expression. In addition, VJ treatment induces apoptosis in colon cancer cells as well as AKT overexpressing cells. Intraperitoneal administration of VJ in pCMV/HCT116 and AKT/HCT-116 xenograft mice revealed decrease in tumor volume in comparison with control mice. We also examined the in vivo efficacy of VJ in ApcMin/+ mouse model. Treatment of ApcMin/+ with VJ (0.5 mg/kg/IP/twice a week) over 12 weeks significantly reduced the number of intestinal polyps (distal 84%; Middle 63% and Proximal 2%) as compared vehicle treated mice. In addition, we also observed 50% reduction in number of colon tumors in VJ treated mice. Conclusion: In conclusion, our studies suggest that VJ inhibits both AKT and Notch1 signaling and induces apoptosis in colon cancer cells. In vivo studies revealed significant reduction in tumor formation in mice models of colon cancer. Hence VJ could be a viable therapeutic agent for treating patients with metastatic colon cancer. Citation Format: Deeksha Pal, Balaji Chandrasekaran, Ashish Tyagi, Becca Von Baby, Houda Alatassi, Murali Ankem, Chendil Damodaran. Chemoprevention of metastatic colon cancer by a novel small molecule [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3930.