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Background Colorectal cancer is the 2nd leading cause of cancer death in Canada. In rapidly dividing tissues such as the intestine or colon, only long-lived, multipotent, self-renewing tissue stem cells have longevity to accumulate mutations and serve as the cellular origin of cancer. In the small intestine, genetic fate mapping studies have demonstrated that there are at least two principal stem cell pools: actively cycling, crypt base cells expressing Lgr5, and quiescent cells situated above the crypt base. Clevers and colleagues have previously shown that Lgr5-expressing cells can give rise to cancer upon mutation. Interestingly, when Lgr5+ stem cells are selectively “killed”, intestinal integrity remains intact and other stem cells restore homeostasis. To determine whether another stem cell population can give rise to cancer in the colon, we examined whether the atypical homeobox protein Hopx, marks stem cells in the colon and whether these cells can give rise to colon cancer. Aims In the present study, we aim to determine whether Hopx-expressing cells are colonic stem cells that contribute to gut healing and can give rise to colonic tumours following the loss of APC. Methods To determine whether Hopx expressing cells show stemness, we crossed Hopx-CreERT mice to R26-TdTomato reporter mice. We then conducted genetic lineage tracing studies in the colon during homeostasis and following dextran sodium sulphate (DSS)-induced colitis. To test the function of Hopx expressing cells, Hopx-CreERT mice were also crossed to R26DTR mice and treated with diphtheria toxin (DT) following tamoxifen. These mice were then exposed to either normal drinking water or DSS to determine the role of Hopx+ cells in colonic regeneration. To test whether Hopx expressing cells can serve as a cellular origin for colon cancer, Hopx-CreERT mice were crossed to Apcf/f (floxed) mice. Results Consistent with the labeling of a stem cell, following tamoxifen, Hopx+ cells expressing tdTomato expanded to lineage trace full colonic crypts within 7 days, and labelling was persistent for greater than 6 months. Interestingly, ablation of Hopx+ cells with DT did not alter weight, histological damage or survival during normal homeostasis, however, Hopx+ cell ablation in mice treated with DSS resulted in increased histological damage. Surprisingly, loss of APC in Hopx-expressing cells did not induce colonic adenomas even after 8 months following tamoxifen administration. Conclusions These findings prove that Hopx expressing cells identify a novel colonic stem cell pool that is redundant in homeostasis, but in the context of injury, are essential for epithelial regeneration. Interestingly, Hopx+ cells do not have the capacity to give rise to colorectal adenomas upon loss of the APC gene. Funding Agencies CIHR

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer death in Canada, with the major risk factor being chronic inflammation. As such, patients with inflammatory bowel disease (IBD) are at an increased risk of CRC. Despite the clear association between inflammation and cancer, the mechanism by which colitis leads to CRC is still not well understood. Our recent work has focused on a colonic epithelial cell known as the tuft cell that uniquely expresses the protein doublecortin-like kinase 1 (Dclk1). Using Cre-dependent lineage tracing of Dclk1-expressing cells, we showed that Dclk1 labels long-lived quiescent cells in the colon that serve as a cellular origin of CRC upon colonic inflammation. AIMS: In this study, we aim to explore the mechanism by which inflammation contributes to tuft cell cancer initiation. We hypothesized that colonic inflammatory insult leads to dedifferentiation of Dclk1+ tuft cells to a stem cell state susceptible to tumor initiation. METHODS: To generate tamoxifen-inducible Cre transgenic mice that allow for Dclk1(+) cell lineage tracing and cell-specific knock-out of the tumor suppressor adenomatous polyposis coli (APC), we first crossed our transgenic Dclk1-CreER(T2) mice to both ROSA26-tdTomato and APC(fl/fl) mice (Dclk1/APC(fl/fl)). To examine the role of dedifferentiation in colonic tumor initiation, these mice were further crossed to Lgr5-DTR-eGFP mice (Lgr5(DTR);Dclk1/APC(fl/fl)). These mice were given tamoxifen and DSS in order to induce tumorigenesis. Mice were administered diphtheria toxin (DT) for six weeks post DSS injury to ablate Lgr5-expressing cells and were compared to Lgr5(DTR)-negative control mice (Dclk1/APC(fl/fl)). RESULTS: Reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA levels revealed significantly reduced Lgr5, and increased RSPO1 and RSPO3 levels in colonic tissues of DSS-administered mice. Ablation of Lgr5(+) cells post DSS-colitis significantly reduced the number of colonic tumors in our Dclk1/APC(fl/fl) mouse model, with no significant difference in tumor size. Lgr5-expressing cells were readily seen throughout colonic tumors arising from Dclk1(+) cells. Interestingly, two weeks post DSS-induced colitis, rare Dclk1(+) cells that co-expressed Lgr5 were also detected. CONCLUSIONS: Our data prove that upon DSS-induced colonic injury, Dclk1(+) tuft cells express the stem cell marker Lgr5 prior to initiation of colonic tumorigenesis. These data suggest that dedifferentiation of Dclk1(+) cells plays an important role in colitis-associated CRC and provide insight into the molecular mechanism by which Dclk1-derived colonic tumors arise. FUNDING AGENCIES: CAG, CIHRCRS

Background: Some trial have demonstrated a benefit of adjuvant fluoropirimidine with or without platinum compounds compared with surgery alone. ITACA-S study was designed to evaluate whether a sequential treatment of FOLFIRI [irinotecan plus 5-fluorouracil/folinic acid (5-FU/LV)] followed by docetaxel plus cisplatin improves disease-free survival in comparison with 5-FU/LV in patients with radically resected gastric cancer. Patients and methods: Patients with resectable adenocarcinoma of the stomach or gastroesophageal junction were randomly assigned to either FOLFIRI (irinotecan 180 mg/m 2 day 1, LV 100 mg/m 2 as 2 h infusion and 5-FU 400 mg/m 2 as bolus, days 1 and 2 followed by 600 mg/m 2 /day as 22 h continuous infusion, q14 for four cycles) followed by docetaxel 75 mg/m 2 day 1, cisplatin 75 mg/m 2 day 1, q21 for three cycles (sequential arm) or De Gramont regimen (5-FU/LV arm). Results: From February 2005 to August 2009, 1106 patients were enrolled, and 1100 included in the analysis: 562 in the sequential arm and 538 in the 5-FU/LV arm. With a median follow-up of 57.4 months, 581 patients recurred or died (297 sequential arm and 284 5-FU/LV arm), and 483 died (243 and 240, respectively). No statistically significant difference was detected for both disease-free [hazard ratio (HR) 1.00; 95% confidence interval (CI): 0.85–1.17; P= 0.974] and overall survival (OS) (HR 0.98; 95% CI: 0.82–1.18; P= 0.865). Five-year disease-free and OS rates were 44.6% and 44.6%, 51.0% and 50.6% in the sequential and 5-FU/LV arm, respectively. Conclusions: A more intensive regimen failed to show any benefit in disease-free and OS versus monotherapy. Clinical trial registration: ClinicalTrials.gov Identifier: NCT01640782.

Recent genomics analysis of the high-grade serous subtype of epithelial ovarian cancer (EOC) show aberrations in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway that result in upregulated signaling activity. Thus, the PI3K/AKT pathway represents a potential therapeutic target for aggressive high-grade EOC. We previously demonstrated that treatment of malignant ascites-derived primary human EOC cells and ovarian cancer cell lines with the allosteric AKT inhibitor Akti-1/2 induces a dormancy-like cytostatic response but does not reduce cell viability. In this report, we show that allosteric AKT inhibition in these cells induces cytoprotective autophagy. Inhibition of autophagy using chloroquine (CQ) alone or in combination with Akti-1/2 leads to a significant decrease in viable cell number. In fact, Akti-1/2 sensitizes EOC cells to CQ-induced cell death by exhibiting markedly reduced EC50 values in combination-treated cells compared with CQ alone. In addition, we evaluated the effects of the novel specific and potent autophagy inhibitor-1 (Spautin-1) and demonstrate that Spautin-1 inhibits autophagy in a Beclin-1-independent manner in primary EOC cells and cell lines. Multicellular EOC spheroids are highly sensitive to Akti-1/2 and CQ/Spautin-1 cotreatments, but resistant to each agent alone. Indeed, combination index analysis revealed strong synergy between Akti-1/2 and Spautin-1 when both agents were used to affect cell viability; Akti-1/2 and CQ cotreatment also displayed synergy in most samples. Taken together, we propose that combination AKT inhibition and autophagy blockade would prove efficacious to reduce residual EOC cells for supplying ovarian cancer recurrence. © The Author 2014. Published by Oxford University Press. All rights reserved.