Christopher Rice

In Vivo Detection of Human TRPV6-Rich Tumors with Anti-Cancer Peptides Derived from Soricidin

Soricidin is a 54-amino acid peptide found in the paralytic venom of the northern short-tailed shrew (Blarina brevicauda) and has been found to inhibit the transient receptor potential of vallinoid type 6 (TRPV6) calcium channels. We report that two shorter peptides, SOR-C13 and SOR-C27, derived from the C-terminus of soricidin, are high-affinity antagonists of human TRPV6 channels that are up-regulated in a number of cancers. Herein, we report molecular imaging methods that demonstrate the in vivo diagnostic potential of SOR-C13 and SOR-C27 to target tumor sites in mice bearing ovarian or prostate tumors. Our results suggest that these novel peptides may provide an avenue to deliver diagnostic and therapeutic reagents directly to TRPV6-rich tumors and, as such, have potential applications for a range of carcinomas including ovarian, breast, thyroid, prostate and colon, as well as certain leukemias and lymphomas.

Nutritional and metabolic requirements for the infection of HeLa cells by Salmonella enterica serovar Typhimurium

Salmonella is the causative agent of a spectrum of human and animal diseases ranging from gastroenteritis to typhoid fever. It is a food - and water - borne pathogen and infects via ingestion followed by invasion of intestinal epithelial cells and phagocytic cells. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted the key glycolytic genes, pfkA and pfkB to show that S. Typhimurium utilizes glycolysis for replication within HeLa cells; however, glycolysis was not absolutely essential for intracellular replication. Using S. Typhimurium strains deleted for genes encoding components of the phosphotransferase system and glucose transport, we show that glucose is a major substrate required for the intracellular replication of S. Typhimurium in HeLa cells. We also deleted genes encoding enzymes involved in the utilization of gluconeogenic substrates and the glyoxylate shunt and show that neither of these pathways were required for intracellular replication of S. Typhimurium within HeLa cells.

From antiangiogenesis to hypoxia: current research and future directions

Angiogenesis has long been recognized as an essential element in tumor growth. Since the conception of antiangiogenesis for cancer therapeutics, great strides have been made in understanding the molecular biology underlying angiogenesis, both in cancer and in physiology. By capitalizing on these advancements through bench-to-bedside research, potent antiangiogenic agents have been developed and tested. To date, the clinical results of most of these antiangiogenic agents have not met expectations. Even with the most successful agents, such as bevacizumab, used either as single agents or in combination with chemotherapy, gains in overall survival of cancer patients have been modest in most cases. In this article, the authors present the evolving views of antiangiogenic therapy, review recent experimental and clinical studies on antiangiogenesis, and address the fundamental role of hypoxia in tumor progression, which may be key to improving the efficacy of antiangiogenic therapy.

Intermittent Induction of HIF-1α Produces Lasting Effects on Malignant Progression Independent of Its Continued Expression

Dysregulation of hypoxia-inducible transcription factors HIF-1α and HIF-2α correlates with poor prognosis in human cancers; yet, divergent and sometimes opposing activities of these factors in cancer biology have been observed. Adding to this complexity is that HIF-1α apparently possesses tumor-suppressing activities, as indicated by the loss-of-function mutations or even homozygous deletion of HIF1A in certain human cancers. As a step towards understanding this complexity, we employed 8-week intermittent induction of a stable HIF-1α variant, HIF1α(PP), in various cancer cell lines and examined the effects on malignant progression in xenografts of immunocompromised mice in comparison to those of HIF2α(PP). Although 8-week treatment led to eventual loss of HIF1α(PP) expression, treated osteosarcoma U-2 OS cells acquired tumorigenicity in the subcutaneous tissue. Furthermore, the prior treatment resulted in widespread invasion of malignant glioma U-87 MG cells in the mouse brain and sustained growth of U-118 MG glioma cells. The lasting effects of HIF-1α on malignant progression are specific because neither HIF2α(PP) nor β-galactosidase yielded similar effects. By contrast, transient expression of HIF1α(PP) in U-87 MG cells or constitutive expression of HIF1α(PP) but not HIF2α(PP) in a patient-derived glioma sphere culture inhibited tumor growth and spread. Our results indicate that intermittent induction of HIF-1α produces lasting effects on malignant progression even at its own expense.

Inhibition of Transient Receptor Potential Vanilloid 6 channel, elevated in human ovarian cancers, reduces tumour growth in a xenograft model

Background: Transient Receptor Potential Vanilloid 6 (TRPV6), a non-voltage gated calcium channel, is implicated in malignancies and correlates with Gleason scores in prostate cancer and with poor prognosis in breast cancer. Data on the TRPV6 status of ovarian malignancies has not received significant attention. The effect of inhibiting TRPV6 activity on ovarian tumour growth has never been reported. Methods: We quantified TRPV6 mRNA and protein in biopsies of five types of ovarian cancer at different stages and grades by quantitative PCR and immunohistochemistry respectively. We verified the presence of TRPV6 in SKOV-3 cells and xenografts by Western Blotting. NOD/SCID mice bearing xenografted ovarian tumours derived from SKOV-3 were treated daily with TRPV6-antagonistic peptides (SOR-C13 and SOR-C27) at 400, 600 and 800 mg/kg delivered intraperitoneally (i.p.) over 12 days. Data from qPCR and tumour growth experiments were compared with a Students t-test. Immunohistochemical ranking of staining were compared with Kruskall-Wallace one-way ANOVA and Dunns Multiple Comparison post-test. Results: TRPV6 mRNA and protein are significantly elevated at all stages and grades of 5 ovarian cancer types over normal tissue. Overall qPCR log2 values (n, mean, ± SEM) for mRNA in tumour (n = 165, 5.06 ± 0.16) were greater (p < 0.05) than normal tissues (n = 26, 0.45 ± 0.41). All stages and grades included in the biopsy arrays were significantly greater than normal tissues. Immunohistochemical staining of TRPV6 was ranked >2 (faint in most cells) in 80.5% of tumours (123) while 92% of normal tissues (23) ranked ≤ 2. Daily i.p. injection with SOR-C13 (400, 600 and 800 mg/kg) over 12 days inhibits tumour growth (59%) at the highest dose compared to non-treated controls. SOR-C27 at 800 mg/kg SOR-C27 inhibited tumour growth 55% after 12 days. Results of daily and intermittent dosing (Days 1, 2, 3 and 8, 9, 10) with SOR-C13 were indistinguishable. Conclusion: TRPV6 mRNA and protein are elevated in biopsies of ovarian cancers compared to normal tissue. Inhibition of TRPV6 activity significantly reduces ovarian tumour growth providing evidence that TRPV6 is a feasible oncology target in ovarian cancers.

Abstract 2891: Molecular profiling of hormone-resistant prostate cancer cells with TRPV6 oncochannel knockout/knockdown

TRPV6 calcium channel is a recognized oncochannel over-expressed in a number of epithelial cancers (e.g. breast, ovarian, prostate). TRPV6, a member of one of seven subfamilies of TRP channels, plays an important role in Ca2+ absorption from the intestinal lumen. TRPV6 over-expression is associated with a poor prognosis particularly with breast and prostate cancers. Although some TRPV6 mechanistic work is available and indicates that NFAT is involved, there is a need to better understand pathways involved in its mechanism of action (MOA). These data could allow the identification of clinically relevant biomarkers and identify potential for synergy of TRPV6-targeting therapies with other anti-cancer treatments. To determine which key pathways TRPV6 influences in prostate cancer, two TRPV6 knockouts (KO) were produced in a castration-resistant prostate cancer cell line (PC-3) using the CRISPR-Cas9 approach with two different gRNAs. In addition, knockdowns (KD) were produced by transfecting PC-3 cells with two TRPV6 siRNA targeting different regions of TRPV6 mRNA. The mRNA expression of 184 genes was analyzed using an RT-qPCR TaqMan array. The panel consisted of genes involved in cancer calcium signalling, genes that have been associated with the MOA of TRPV6 and genes directly or indirectly involved in NFAT signalling. Gene profiling results were generally consistent between the TRPV6 KD and KO cells. Results showed a dramatic reduction in expression of TRPV6 mRNA in the KO/KD cells (14 to 166-fold). TRPV6 KO/KD affected multiple genes involved in cancer cell proliferation (e.g. ESR1, CDH1), metastasis (e.g. SNAI1, MUC1/16), resistance to apoptosis (e.g. Bcl-2-type proteins, Fos) and angiogenesis (e.g. VEGFA/B, FLT4), as well as genes involved in the M2 immune tumor microenvironment (e.g. IL6 and VEGFA). The TRPV6 KO/KD data confirm the basic MOA of TRPV6, activating NFAT by increasing cytosolic calcium and provide information on the downstream pathways involved (e.g. NF-kB, estrogen and MAPK). The data allow for the identification of clinical efficacy biomarkers for TRPV6 inhibitors (e.g. a panel of CXCL12, FLT4, MUC16 and IL-6). Furthermore, results indicate TRPV6 inhibitors have the potential to modify the immune composition of the tumor microenvironment from a tumor promoting to a tumor control response, and thus may help trigger an anti-cancer immune defence. This study demonstrates a central role of TRPV6 and calcium signalling in cancer development. Citation Format: Tyler Lutes, Michelle Davey, Christopher Rice, Tyson MacCormack, John M. Stewart, Dominique Dugourd. Molecular profiling of hormone-resistant prostate cancer cells with TRPV6 oncochannel knockout/knockdown [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 2891.

Abstract LB-128: High prevalence of elevated TRPV6 mRNA in pancreatic ductal adenocarcinoma

SOR-C13, a 13-mer peptide derived from soricidin, the paralytic protein component of saliva of the Northern Short-tailed shrew, just completed an open-label, all comers phase I clinical trial for the treatment of epithelial-derived cancers. SOR-C13 is a first-in-class drug candidate that specifically targets and inhibits the TRPV6 calcium channel - a recognized oncochannel over-expressed in a number of epithelial cancers (e.g. breast, ovarian, prostate). TRPV6 over-expression is associated with a poor prognosis particularly with breast and prostate cancers. SOR-C13 is the first TRPV6-targeting drug to enter clinical development. Pancreatic patients were enrolled in the phase I clinical trial for SOR-C13 and for this reason the TRPV6 gene expression in pancreatic tumour biopsies was assessed. Association of TRPV6 oncochannel expression with aggressive pancreatic adenocarcinoma compared to neuroendocrine tumours has never been investigated. Pancreatic adenocarcinoma represents 90% of the pancreatic tumours and is associated with a poor prognosis compared to neuroendocrine tumours. TRPV6 gene expression was assessed in 19 pancreatic tumor biopsies including five adenocarcinoma (Grade I to III), one acinar cell (exocrine), and 13 neuroendocrine (Islet cell) tumors by two-step RT-qPCR. TRPV6 and β-actin (reference gene) mRNA expression was evaluated in duplicate in a reproducible TRPV6 plus β-actin duplex two-step RT-qPCR assay. The acinar cell, and 69% (9/13) of the neuroendocrine tumors had no detectable TRPV6 mRNA expression with >90% of neuroendocrine tumors (12/13) having very little TRPV6 mRNA expression ( Citation Format: Tyler Lutes, Dominique Dugourd, Michelle Davey, Christopher Rice, Stephanie St-Pierre, Jack M. Stewart. High prevalence of elevated TRPV6 mRNA in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-128.

Abstract LB-051: Cancer cell binding, internalization and intracellular localization of a novel therapeutic peptide SOR-C13 targeting the TRPV6 oncochannel

SOR-C13, a 13-mer peptide derived from soricidin, the paralytic protein component of saliva of the Northern Short-tailed shrew, has recently completed a phase I clinical trial for the treatment of epithelial-derived cancers. SOR-C13 specifically targets and inhibits the TRPV6 calcium channel - a recognized oncochannel over-expressed in a number of epithelial cancers (e.g. breast, ovarian, prostate). SOR-C13 is the first TRPV6-targeting drug to enter clinical development. Xenograft and fluorescent imaging studies provide in vivo evidence of SOR-C13 efficacy in a range of epithelial tumors as well as SOR-C139s preferential accumulation at tumour sites. To demonstrate the specificity of the peptide for TRPV6 and determine its fate, modified SOR-C13 peptide was labeled with fluorescein to enable monitoring of cellular binding and internalization in high versus low TRPV6 cancer cell lines. Various organelle-specific fluorescent trackers for nuclei, actins, lysosomes, and endoplasmic reticula were paired with labeled SOR-C13 peptides and anti-TRPV6 antibody in fluorescence imaging studies to monitor intracellular trafficking. Rapid peptide binding and internalization was observed on incubation of fluorescently labeled SOR-C13 with a breast cancer cell line (T-47D) expressing high levels of the ion channel. Significantly less SOR-C13 binding was observed in an ovarian cancer cell line (SKOV-3) with low TRPV6 levels. Fluorescently labeled SOR-C13 scramble peptide was included as a control and did not bind to high TRPV6 cells. Co-localization studies did not demonstrate a clear association of the fluorescently labeled SOR-C13 with lysosomes, the ER or actin. This study demonstrates that SOR-C13 not only specifically binds to TRPV6-expressing cancer cells according to their level of TRPV6 expression but is also internalized. The correlation of TRPV6 expression in tumors and clinical efficacy will be evaluated during the next SOR-C13 clinical trials. Additionally, these results set the stage for further development of SOR-C13-based peptide drug conjugates for TRPV6 targeted anti-cancer therapies. Citation Format: Michelle Davey, Dominique Dugourd, Tyler Lutes, Christopher Rice, Sean Gormley, Stephanie St. Pierre, John M. Stewart. Cancer cell binding, internalization and intracellular localization of a novel therapeutic peptide SOR-C13 targeting the TRPV6 oncochannel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-051.

Abstract CT142: A phase I open-label, dose escalation study of a novel peptide (SOR-C13) antagonistic to the TRPV6 ion channel in patients with advanced solid tumor cancers

Background: SOR-C13 is a first-in-man selective peptide inhibitor of Transient Receptor Potential Vanilloid 6 (TRPV6) calcium oncochannel. TRPV6 is highly expressed in carcinomas including prostate, breast, lung and ovary and expression is correlated with poor outcomes. TRPV6-mediated Ca2+ entry is responsible for maintaining a high proliferation rate, increasing cell survival and apoptosis resistance. Since SOR-C13 blocks TRPV6-mediated Ca2+ influx it was evaluated as a single agent in patients with late-stage carcinoma. Methods: This was a phase I, multi-center, open-label, dose escalation study to assess safety and tolerability of SOR-C13 in subjects with advanced carcinomas commonly known to express the TRPV6 channel (NCT01578564). Subjects received two 21-day cycles of treatment consisting of SOR-C13 for 3 d on/4 d off, 3 d on/11 d off (21 d/cycle). Tested doses were 1.375, 2.75, 4.13 and 6.2 mg/kg given as i.v. infusion initially over 20 m moving to 90 m. In the event of clinically meaningful response (tumor response or stable disease), additional cycles were offered to subjects. Subjects were assessed for safety, tolerability and preliminary efficacy. Results: Twenty-three subjects with advanced refractory solid tumors were in the study. No study drug-related serious adverse events occurred during the study. The most frequently reported treatment-emergent adverse events (TEAEs) were fatigue (30%), hypoalbuminemia (30%), anemia (30%), urinary tract infection (30%), blood calcium decreased (22%), decreased appetite (22%), nausea (22%), constipation (17%), aspartate aminotransferase increased (17%), cough (17%), blood alkaline phosphatase increased (13%), diarrhea (13%), hypercalcemia (13%), hyperkalemia (13%), hypocalcemia (13%). Anemia (Grade 1 to 3) was the only TEAE reflecting clinically significant hematological abnormalities. In all but one case, these events were assessed as unrelated or unlikely related to the study drug. The only safety concern identified definitely related to SOR-C13 infusion was reductions of serum calcium that were short-lived (resolved during the post-infusion observation time (4 h or within Conclusions: SOR-C13 was safe and well tolerated in subjects with advanced solid tumors of epithelial origin and demonstrates potential activity in patients with advanced solid tumor cancer. Citation Format: Siqing Fu, Hal H. Hirte, Stephen Welch, Toney T. Ilenchuk, Dominique Dugourd, Tyler Lutes, Christopher Rice, Jack M. Stewart. A phase I open-label, dose escalation study of a novel peptide (SOR-C13) antagonistic to the TRPV6 ion channel in patients with advanced solid tumor cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT142.

Abstract B83: Targeting TRPV6 oncochannel for the treatment of pancreatic cancer: A Phase I trial experience

Background: SOR-C13 is a first-in-human selective peptide inhibitor of Transient Receptor Potential Vanilloid 6 (TRPV6) oncochannel. TRPV6 is highly elevated in carcinomas including prostate, breast, lung and ovary and is correlated with poor outcomes. TRPV6-mediated Ca2+ entry is responsible for maintaining a high proliferation rate, increasing cell survival and apoptosis resistance. Since SOR-C13 blocks TRPV6-mediated Ca2+ influx it was evaluated as a single agent in patients with metastatic carcinomas. Methods: This was a Phase I, multi-center, open-label, dose escalation study to assess safety and tolerability of SOR-C13 in subjects with advanced carcinomas commonly known to express the TRPV6 channel (NCT01578564). Subjects received intravenous infusion of SOR-C13 over 90 minutes for 3 d on/4 d off, 3 d on/11 d off (21 d/cycle) until tumor progression, prohibitive toxicity or patient withdrawal. Tumors were assessed according to RECIST 1.1. Results: A total of 23 patients were enrolled to 4 dose levels and 119 cycles of therapy were administered. No study drug-related SAEs occurred. Twelve of 22 evaluable subjects (54.5%) had stable disease after 2 cycles. Promising clinical benefit was observed in two patients with advanced metastatic ductal pancreatic cancer. Subject 304 showed stable disease (SD) at dose level of 2.75 mg/kg for 12 weeks, and was removed from the study for persistent grade 2 hepatic transaminase elevation for more than 21 days. Subject 312, treated at dose level of 6.2 mg/kg, showed 7% decrease in tumor size after 2 cycles and a 27% decrease after 4 cycles. Tumor marker CA 19-9 responses were consistent with most changes in tumor size over the treatment cycles with suggestions of a dose related response and correlation with tumor size. The response of this biomarker was particularly evident in the patient initially treated at 6.2 mg/kg where it decreased by 29% after cycle two and by 55% after cycle 4. This patient was removed from the study after a total of 27 weeks 2 days. Both subjects failed at least three prior regimens of anticancer therapy: Subject 304 (i) IMRT/Tomotherapy plus chemoradiation with Folfirinox, (ii) Carboplatin/gemcitabine/Tarceva (iii) Abraxane/Gemcitabine, (iv) IMRT/Tomotherapy; Subject 312 (i) Folfirinox; (ii) gemcitabine and Abraxane; and, (iii) Xeloda. Conclusions: SOR-C13 was safe and well tolerated in subjects with advanced epithelial malignancies. The clinical benefit observed in patients with metastatic pancreatic cancer, particularly 27% reduction of the sum of tumor diameters after 4 cycles warrants further investigation of this novel treatment in Phase II studies. Citation Format: Siqing Fu, Stephen Welch, Toney T. Ilenchuk, Dominique Dugourd, Tyler Lutes, Chris Rice, Jack M. Stewart.{Authors}. Targeting TRPV6 oncochannel for the treatment of pancreatic cancer: A Phase I trial experience. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B83.