Achinto Saha

Dietary energy balance modulates prostate cancer progression in Hi-Myc mice

Male Hi-Myc mice were placed on three dietary regimens [30% calorie restriction (CR), overweight control (modified AIN76A with 10 kcal% fat), and a diet-induced obesity regimen (DIO) 60 kcal% fat]. All diet groups had approximately similar incidence of hyperplasia and low-grade prostatic intraepithelial neoplasia in the ventral prostate at 3 and 6 months of age. However, 30% CR significantly reduced the incidence of in situ adenocarcinomas at 3 months compared with the DIO group and at 6 months compared with both the overweight control and DIO groups. Furthermore, the DIO regimen significantly increased the incidence of adenocarcinoma with aggressive stromal invasion, as compared with the overweight control group (96% vs. 65%, respectively; P = 0.02) at the 6-month time point. In addition, at both 3 and 6 months, only in situ carcinomas were observed in mice maintained on the 30% CR diet. Relative to overweight control, DIO increased whereas 30% CR reduced activation of Akt, mTORC1, STAT3, and NFκB (p65) in ventral prostate. DIO also significantly increased (and 30% CR decreased) numbers of T-lymphocytes and macrophages in the ventral prostate compared with overweight control. The mRNA levels for interleukin (IL) 1α, IL1β, IL6, IL7, IL23, IL27, NFκB1 (p50), TNFα, and VEGF family members were significantly increased in the ventral prostate of the DIO group compared with both the overweight control and 30% CR diet groups. Collectively, these findings suggest that enhanced growth factor (Akt/mTORC1 and STAT3) and inflammatory (NFκB and cytokines) signaling may play a role in dietary energy balance effects on prostate cancer progression in Hi-Myc mice. Cancer Prev Res; 4(12); 2002–14. ©2011 AACR.

6-Shogaol from Dried Ginger Inhibits Growth of Prostate Cancer Cells Both In Vitro and In Vivo through Inhibition of STAT3 and NF-κB Signaling

Despite much recent progress, prostate cancer continues to represent a major cause of cancer-related mortality and morbidity in men. Prostate cancer is the most common nonskin neoplasm and second leading cause of death in men. 6-Shogaol (6-SHO), a potent bioactive compound in ginger (Zingiber officinale Roscoe), has been shown to possess anti-inflammatory and anticancer activity. In the present study, the effect of 6-SHO on the growth of prostate cancer cells was investigated. 6-SHO effectively reduced survival and induced apoptosis of cultured human (LNCaP, DU145, and PC3) and mouse (HMVP2) prostate cancer cells. Mechanistic studies revealed that 6-SHO reduced constitutive and interleukin (IL)-6–induced STAT3 activation and inhibited both constitutive and TNF-α–induced NF-κB activity in these cells. In addition, 6-SHO decreased the level of several STAT3 and NF-κB–regulated target genes at the protein level, including cyclin D1, survivin, and cMyc and modulated mRNA levels of chemokine, cytokine, cell cycle, and apoptosis regulatory genes (IL-7, CCL5, BAX, BCL2, p21, and p27). 6-SHO was more effective than two other compounds found in ginger, 6-gingerol, and 6-paradol at reducing survival of prostate cancer cells and reducing STAT3 and NF-κB signaling. 6-SHO also showed significant tumor growth inhibitory activity in an allograft model using HMVP2 cells. Overall, the current results suggest that 6-SHO may have potential as a chemopreventive and/or therapeutic agent for prostate cancer and that further study of this compound is warranted. Cancer Prev Res; 7(6); 627–38. ©2014 AACR.

Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth

Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. Under conditions of elevated ROS, endogenous L-cysteine (L-Cys) production is insufficient for GSH synthesis. This necessitates uptake of L-Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(−) transporter. We show that administration of an engineered and pharmacologically optimized human cyst(e)inase enzyme mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet this treatment results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in both prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53−/− mice, which develop disease resembling human chronic lymphocytic leukemia. It was observed that enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods, suggesting that cyst(e)inase represents a safe and effective therapeutic modality for inactivating antioxidant cellular responses in a wide range of malignancies.Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than non-malignant cells due to genetic alterations and abnormal growth and as a result, maintenance of the anti-oxidant glutathione (GSH) is essential for their survival and proliferation1–3. Under elevated ROS conditions endogenous l-Cysteine (l-Cys) production is insufficient for GSH synthesis, necessitating l-Cys uptake, predominantly in its disulfide form l-Cystine (CSSC) via the xCT(−) transporter. Here we show that administration of an engineered, pharmacologically optimized, human Cyst(e)inase enzyme mediates sustained depletion of the extracellular l-Cys and CSSC pool in mice and non-human primates, selectively causes cell cycle arrest and death (PI and Annexin-V staining) in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS, yet results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53−/− mice that develop disease resembling human chronic lymphocytic leukemia. The observation that enzyme-mediated depletion of the serum l-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods suggests that Cyst(e)inase represents a safe and effective therapeutic modality for inactivating anti-oxidant cellular responses in a wide range of malignancies4,5.

Effect of Metformin, Rapamycin, and Their Combination on Growth and Progression of Prostate Tumors in HiMyc Mice

In this study, we compared the effect of oral administration of metformin (MET) and rapamycin (RAPA) alone or in combination on prostate cancer development and progression in HiMyc mice. MET (250 mg/kg body weight in the drinking water), RAPA (2.24 mg/kg body weight microencapsulated in the diet), and the combination inhibited progression of prostatic intraepithelial neoplasia lesions to adenocarcinomas in the ventral prostate (VP). RAPA and the combination were more effective than MET at the doses used. Inhibition of prostate cancer progression in HiMyc mice by RAPA was associated with a significant reduction in mTORC1 signaling that was further potentiated by the combination of MET and RAPA. In contrast, treatment with MET alone enhanced AMPK activation, but had little or no effect on mTORC1 signaling pathways in the VP of HiMyc mice. Further analyses revealed a significant effect of all treatments on prostate tissue inflammation as assessed by analysis of the expression of cytokines, the presence of inflammatory cells and NFκB signaling. MET at the dose used appeared to reduce prostate cancer progression primarily by reducing tissue inflammation whereas RAPA and the combination appeared to inhibit prostate cancer progression in this mouse model via the combined effects on both mTORC1 signaling as well as on tissue inflammation. Overall, these data support the hypothesis that blocking mTORC1 signaling and/or tissue inflammation can effectively inhibit prostate cancer progression in a relevant mouse model of human prostate cancer. Furthermore, combinatorial approaches that target both pathways may be highly effective for prevention of prostate cancer progression in men. Cancer Prev Res; 8(7); 597–606. ©2015 AACR.

Proinflammatory CXCL12–CXCR4/CXCR7 Signaling Axis Drives Myc-Induced Prostate Cancer in Obese Mice

Obesity is a prognostic risk factor in the progression of prostate cancer; however, the molecular mechanisms involved are unclear. In this study, we provide preclinical proof of concept for the role of a proinflammatory CXCL12-CXCR4/CXCR7 signaling axis in an obesity-driven mouse model of myc-induced prostate cancer. Analysis of the stromal vascular fraction from periprostatic white adipose tissue from obese HiMyc mice at 6 months of age revealed a dramatic increase in mRNAs encoding various chemokines, cytokines, growth factors, and angiogenesis mediators, with CXCL12 among the most significantly upregulated genes. Immunofluorescence staining of ventral prostate tissue from obese HiMyc mice revealed high levels of CXCL12 in the stromal compartment as well as high staining for CXCR4 and CXCR7 in the epithelial compartment of tumors. Prostate cancer cell lines derived from HiMyc tumors (HMVP2 and derivative cell lines) displayed increased protein expression of both CXCR4 and CXCR7 compared with protein lysates from a nontumorigenic prostate epithelial cell line (NMVP cells). CXCL12 treatment stimulated migration and invasion of HMVP2 cells but not NMVP cells. These effects of CXCL12 on HMVP2 cells were inhibited by the CXCR4 antagonist AMD3100 as well as knockdown of either CXCR4 or CXCR7. CXCL12 treatment also produced rapid activation of STAT3, NFκB, and MAPK signaling in HMVP2 cells, which was again attenuated by either AMD3100 or knockdown of CXCR4 or CXCR7. Collectively, these data suggest that CXCL12 secreted by stromal cells activates invasiveness of prostate cancer cells and may play a role in driving tumor progression in obesity. Targeting the CXCL12-CXCR4/CXCR7 axis could lead to novel approaches for offsetting the effects of obesity on prostate cancer progression. Cancer Res; 77(18); 5158-68. ©2017 AACR.

Lin neg Sca-1 high CD49f high prostate cancer cells derived from the Hi-Myc mouse model are tumor-initiating cells with basal-epithelial characteristics and differentiation potential in vitro and in vivo

A cell line was established from ventral prostate (VP) tumors of one-year-old Hi-Myc mice. These cells, called HMVP2 cells, are LinnegSca-1highCD49fhigh with high CD44 and CD29 expression and express CK14, Sca-1 and CD49f (but not CK8), suggesting basal-epithelial characteristics. Furthermore, HMVP2 cells form spheroids and both the cells and spheroids produce tumors in syngeneic mice. After four days of culture, HMVP2 spheroids underwent a gradual transition from LinnegSca-1highCD49fhigh expression to LinnegSca-1lowCD49flow while a subpopulation of the cells retained the original LinnegSca-1highCD49fhigh expression pattern. Additional cell subpopulations expressing Lin positive markers were also present suggesting further differentiation of HMVP2 spheroids. Two additional highly tumorigenic cell lines (HMVP2A1 and HMVP2A2) were isolated from HMVP2 cells after subsequent tumor formation in FVB/N mice. Concurrently, we also established cell lines from the VP of 6 months old Hi-Myc mice (named as HMVP1) and FVB/N mice (called NMVP) having less aggressive growth properties compared to the other three cell lines. AR expression was reduced in HMVP2 cells compared to NMVP and HMVP1 cells and almost absent in HMVP2A1 and HMVP2A2 cells. These cell lines will provide valuable tools for further mechanistic studies as well as preclinical studies to evaluate preventive and/or therapeutic agents for prostate cancer.

Combinatorial treatment with natural compounds in prostate cancer inhibits prostate tumor growth and leads to key modulations of cancer cell metabolism

High-throughput screening of a natural compound library was performed to identify the most efficacious combinatorial treatment on prostate cancer. Ursolic acid, curcumin and resveratrol were selected for further analyses and administered in vivo via the diet, either alone or in combination, in a mouse allograft model of prostate cancer. All possible combinations of these natural compounds produced synergistic effects on tumor size and weight, as predicted in the screens. A subsequent untargeted metabolomics and metabolic flux analysis using isotopically labeled glutamine indicated that the compound combinations modulated glutamine metabolism. In addition, ASCT2 levels and STAT3, mTORC1 and AMPK activity were modulated to a greater extent by the combinations compared to the individual compounds. Overall, this approach can be useful for identifying synergistic combinations of natural compounds for chemopreventive and therapeutic interventions.Prostate cancer: Combination of natural compounds limits tumor growthCombinations of two molecules found naturally in edible plants synergistically help reduce tumor growth in a mouse model of prostate cancer. Stefano Tiziani and John DiGiovanni from the University of Texas at Austin, and colleagues screened a library of 142 natural compounds for the effects of each molecule, alone or in combination, on the viability of cells from mouse and human prostate cancer cell lines. Amongst other promising combinations, the researchers identified ursolic acid and curcumin as the most promising combination for inhibiting tumor growth. (These compounds are found naturally in apple peels and turmeric, respectively.) In mice with implanted prostate tumors, the two compounds synergistically reduced tumor volume and weight, while in cell culture the researchers showed that the compound-combination strategy modulated metabolism of a critical amino acid and other cell signaling pathways.

Abstract 2132: Novel therapeutic approach through systemic depletion of L-cyst(e)ine with engineered cyst(e)inase enzyme for suppression of prostate tumor growth

Prostate cancer (PCa) is the most common non-skin neoplasm and second leading cause of cancer death in men in the USA. The major drawbacks of PCa treatment is the development of resistance to androgen ablation therapy. Due to abnormal growth and genetic alterations, cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than the normal cells. The L-cysteine (Cys) containing tripeptide, glutathione (GSH) is the major intracellular antioxidant and is essential for the survival and proliferation of cancer cells. Under conditions of elevated ROS, endogenous Cys production is insufficient for GSH synthesis. This necessitates uptake of Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(-) transporter. Cys is a non-essential amino acid in animals; therefore, eliminating Cys and CSSC uptake should selectively impact tumors that display increased ROS production, without causing an adverse effect on normal physiology. However, inhibition of xCT(-) alone is insufficient because free Cys is still imported via other transporters. A superior approach is the elimination of Cys and CSSC through the action of an enzyme that converts these amino acids into non-toxic products. Based on the idea that enzyme mediated systemic depletion of the serum Cys/CSSC pool would constitute a powerful and completely novel therapeutic approach, we developed a genetically engineered and pharmacologically optimized human enzyme called cyst(e)inase. We show that administration of cyst(e)inase mediates sustained depletion of the extracellular Cys and CSSC pool in mice. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts and reduced tumor growth in PCa xenografts. Mechanistically, cyst(e)inase treatment increased AMPK phosphorylation, reduced mTORC1 activity, formation of LC3 II as well as modulation of several cell cycle proteins including p27, c-Myc, CDK2, CDK4, pRB, E2F4 and cyclins A, D1 and E1. Further studies showed cyst(e)inase produced synergistic effects with a GSH synthesis inhibitor, buthionine sulfoximine and the natural compound, curcumin for cell growth inhibition and ROS production in vitro. Cyst(e)inase also showed synergistic tumor growth inhibition with curcumin in a xenograft model of human castrate resistant PCa cells. Collectively, enzyme-mediated depletion of serum Cys and CSSC pool suppresses the growth of prostate tumors, and is very well tolerated. These results suggest that cyst(e)inase represents a potentially safe and effective therapeutic modality as a single agent or in combination for the treatment of prostate and possibly other cancers. Citation Format: Achinto Saha, Shira L. Cramer, Sabin Kshattry, Stefano Tiziani, Everett Stone, George Georgiou, John DiGiovanni. Novel therapeutic approach through systemic depletion of L-cyst(e)ine with engineered cyst(e)inase enzyme for suppression of prostate tumor growth [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 2132. doi:10.1158/1538-7445.AM2017-2132

Obesity, Inflammation, and Prostate Cancer

Obesity and the associated metabolic syndrome produce a complex set of alterations both systemically and locally in tissues that support cancer development and progression. In prostate cancer (PCa), the weight of evidence suggests that obesity is primarily associated with more aggressive disease and increased risk of biochemical failure following prostatectomy or radiation treatment. Inflammation processes and inflammation-associated signaling pathways are upregulated in the obese state, and both human and mouse studies support an important role for inflammation in obesity-driven PCa progression. Inflammation signaling pathways along with other signaling pathways (e.g., growth factor signaling pathways) altered in the obese state represent promising targets for both lifestyle and pharmacologic interventions to prevent or control PCa progression.

Adipose stromal cell targeting suppresses prostate cancer epithelial-mesenchymal transition and chemoresistance

Fat tissue, overgrowing in obesity, promotes the progression of various carcinomas. Clinical and animal model studies indicate that adipose stromal cells (ASC), the progenitors of adipocytes, are recruited by tumors and promote tumor growth as tumor stromal cells. Here, we investigated the role of ASC in cancer chemoresistance and invasiveness, the attributes of tumor aggressiveness. By using human cell co-culture models, we demonstrate that ASC induce epithelial-mesenchymal transition (EMT) in prostate cancer cells. Our results for the first time demonstrate that ASC interaction renders cancer cells more migratory and resistant to docetaxel, cabazitaxel, and cisplatin chemotherapy. To confirm these findings in vivo, we compared cancer aggressiveness in lean and obese mice grafted with prostate tumors. We show that obesity promotes EMT in cancer cells and tumor invasion into the surrounding fat tissue. A hunter-killer peptide D-CAN, previously developed for targeted ASC ablation, suppressed the obesity-associated EMT and cancer progression. Importantly, cisplatin combined with D-CAN was more effective than cisplatin alone in suppressing growth of mouse prostate cancer allografts and xenografts even in non-obese mice. Our data demonstrate that ASC promote tumor aggressiveness and identify them as a target of combination cancer therapy.