SUMMARY We report a paracrine effect whereby endothelial cells (ECs) promote the cancer stem cell (CSC) phenotype of human colorectal cancer (CRC) cells. We showed that, without direct cell-cell contact, ECs secrete factors that promoted the CSC phenotype in CRC cells via Notch activation. In human CRC specimens, CD133 and Notch intracellular domain-positive cells co-localized with CRC cells in perivascular regions. An EC-derived, soluble form of Jagged-1, via ADAM17 proteolytic activity, led to Notch activation in CRC cells in a paracrine manner; these effects were blocked by immunodepletion of Jagged-1 in EC conditioned medium or blockade of ADAM17 activity. ECs play an active role in promoting Notch signaling and the CSC phenotype by secreting soluble Jagged-1.

Altered metabolism in cancer cells is suspected to contribute to chemoresistance but the precise mechanisms are unclear. Here we show that intracellular ATP levels are a core determinant in the development of acquired cross-drug resistance of human colon cancer cells that harbor different genetic backgrounds. Drug-resistant cells were characterized by defective mitochondrial ATP production, elevated aerobic glycolysis, higher absolute levels of intracellular ATP and enhanced HIF-1α-mediated signaling. Interestingly, direct delivery of ATP into cross-chemoresistant cells destabilized HIF-1α and inhibited glycolysis. Thus, drug-resistant cells exhibit a greater “ATP debt” defined as the extra amount of ATP needed to maintain homeostasis of survival pathways under genotoxic stress. Direct delivery of ATP was sufficient to render drug-sensitive cells drug resistant. Conversely, depleting ATP by cell treatment with an inhibitor of glycolysis, 3-bromopyruvate, was sufficient to sensitize cells cross-resistant to multiple chemotherapeutic drugs. In revealing intracellular ATP levels are a core determinant of chemoresistance in colon cancer cells, our findings may offer a foundation for new improvements to colon cancer treatment.

Epithelial–mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT was shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcription factor that mediates EMT in a number of tumor types, including colorectal cancer (CRC). Our study was done to determine the role of Snail in mediating EMT and CSC function in CRC. Human CRC specimens were stained for Snail expression, and human CRC cell lines were transduced with a retroviral Snail construct or vector control. Cell proliferation and chemosensitivity to oxaliplatin of the infected cells were determined by the MTT (colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Migration and invasion were determined in vitro using modified Boyden chamber assays. EMT and putative CSC markers were analyzed using Western blotting. Intravenous injection of tumor cells was done to evaluate their metastatic potential in mice. Snail was overexpressed in human CRC surgical specimens. This overexpression induced EMT and a CSC-like phenotype in human CRC cells and enhanced cell migration and invasion (P < 0.002 vs. control). Snail overexpression also led to an increase in metastasis formation in vivo (P < 0.002 vs. control). Furthermore, the Snail-overexpressing CRC cells were more chemoresistant to oxaliplatin than control cells. Increased Snail expression induces EMT and the CSC-like phenotype in CRC cells, which enhance cancer cell invasion and chemoresistance. Thus, Snail is a potential therapeutic target in metastatic CRC.

Importance Hyperthermic intraperitoneal chemotherapy (HIPEC) has been employed within various multimodality strategies for the prevention and treatment of gastric cancer peritoneal carcinomatosis. Objective To systematically evaluate the role of HIPEC in gastric cancer and clarify its effectiveness at different stages of peritoneal disease progression. Data Sources Medline and Embase databases between January 1, 1985, and June 1, 2016. Study Selection Randomized control trials (RTC) and high-quality nonrandomized control trials (NRCTs) selected on a validated tool (Methodological Index for Nonrandomized Studies) comparing HIPEC and standard oncological management for the treatment of advanced stage gastric cancer with and without peritoneal carcinomatosis were considered. Data Extraction and Synthesis A random-effects network meta-analysis. Main Outcomes and Measures The primary outcomes were overall survival and disease recurrence. Secondary outcomes were overall complications, type of complications, and sites of recurrence. Results A total of 11 RCTs and 21 NRCTs (2520 patients) were included. For patients without the presence of peritoneal carcinomatosis (PC), the overall survival rates between the HIPEC and control groups at 3 or 5 years resulted in favor of the HIPEC group (RR=0.82, P=0.01). No difference in the 3-year overall survival (RR=0.99, P=0.85) in but a prolonged median survival of 4 months in favor of the HIPEC group (WMD=4.04, P<0.001) was seen in patients with PC. HIPEC was associated with significantly higher risk of complications for both patients with PC (RR=2.15, P<0.01) and without (RR=2.17, P<0.01). This increased risk in the HIPEC group was related to systemic drugs toxicity. Anastomotic leakage rates were found to be similar between groups. Conclusions and Relevance Our study demonstrates a survival advantage of the use of HIPEC as a prophylactic strategy and suggests that patients whose disease burden is limited to positive cytology and limited nodal involvement may benefit the most from HIPEC. For patients with extensive carcinomatosis, the completeness of cytoreductive surgery is a critical prognostic factor for survival. Future RCTs should better define patient selection criteria.

Background & Aims Metastatic gastrointestinal neuroendocrine tumors (NETs) are frequently refractory to chemotherapy. Chemoresistance in various malignancies has been attributed to cancer stem cells (CSCs). We sought to identify gastrointestinal neuroendocrine CSCs (N-CSCs) in surgical specimens and a NET cell line and to characterize novel N-CSC therapeutic targets. Methods Human gastrointestinal NETs were evaluated for CSCs using the Aldefluor assay. In vitro sphere-forming assay was performed on primary NET cells. CNDT2.5, a human midgut carcinoid cell line, was used for in vitro (sphere-formation) and in vivo (tumorigenicity assays) CSC studies. N-CSC protein expression was characterized using Western blotting. In vivo, systemic siRNA administration targeted Src. Results Using the Aldefluor assay, aldehyde dehydrogenase–positive (ALDH+) cells comprised 5.8% ± 1.4% (mean ± SEM) of cells from 19 patient samples. Though many primary cell lines failed to grow, CNDT96 ALDH+ cells formed spheres in anchorage-independent conditions, whereas ALDH− cells did not. CNDT2.5 ALDH+ cells formed spheres, whereas ALDH− cells did not. In vivo, ALDH+ CNDT2.5 cells generated more tumors, with shorter latency than ALDH− or sham-sorted cells. Compared with non-CSCs, ALDH+ cells demonstrated increased expression of activated Src, Erk, Akt, and mTOR. In vivo, anti-Src siRNA treatment of ALDH+ tumors reduced tumor mass by 91%. Conclusions CSCs are present in neuroendocrine tumors (NETs), demonstrated by in vitro sphere-formation and in vivo tumorigenicity assays. Src was activated in N-CSCs and represents a potential therapeutic target in gastrointestinal NETs.

Combination chemotherapy is standard for metastatic colorectal cancer (CRC); however, nearly all patients develop drug resistance. Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may enable identification of novel targets and more effective therapy. Irinotecan is commonly used in first- and second-line therapy for patients with metastatic CRC, with the active metabolite being SN38. Emerging evidence suggests that altered metabolism in cancer cells is fundamentally involved in the development of drug resistance. Using Oncomine and unbiased proteomic profiling, we found that ATP citrate lyase (ACLy), the first-step rate-limiting enzyme for de novo lipogenesis, was upregulated in CRC compared to its levels in normal mucosa and in chemoresistant CRC cells compared to isogenic chemo-naïve CRC cells. Overexpression of exogenous ACLy by lentivirus transduction in chemo-naïve CRC cells led to significant chemoresistance to SN38 but not to 5-fluorouracil or oxaliplatin. Knockdown of ACLy by siRNA or inhibition of its activity by a small-molecule inhibitor sensitized chemo-naïve CRC cells to SN38. Furthermore, ACLy was significantly increased in cancer cells that had acquired resistance to SN38. In contrast to chemo-naïve cells, targeting ACLy alone was not effective in re-sensitizing resistant cells to SN38, due to a compensatory activation of the AKT pathway triggered by ACLy suppression. Combined inhibition of AKT signaling and ACLy successfully re-sensitized SN38-resistant cells to SN38. We conclude that targeting ACLy may improve the therapeutic effects of irinotecan and that simultaneous targeting of ACLy and AKT may be warranted to overcome SN38 resistance.

Background:Recent studies suggest that cancer stem cells (CSCs) mediate chemoresistance, but interestingly, only a small percentage of cells in a resistant tumour are CSCs; this suggests that non-CSCs survive by other means. We hypothesised that chemoresistant colorectal cancer (CRC) cells generate soluble factors that enhance survival of chemonaive tumour cells.Methods:Chemoresistant CRC cells were generated by serial passage in oxaliplatin (Ox cells). Conditioned media (CM) was collected from parental and oxaliplatin-resistant (OxR) cells. CRC cells were treated with CM and growth and survival were assessed. Tumour growth rates were determined in nude mice after cells were treated with CM. Mass spectrometry (MS) identified proteins in CM. Reverse phase protein microarray assays determined signalling effects of CM in parental cells.Results:Oxaliplatin-resistant CM increased survival of chemo-naive cells. CSC CM also increased growth of parental cells. Parental and OxR mixed tumours grew larger than tumours composed of parental or OxR cells alone. Mass spectrometry detected unique survival-promoting factors in OxR CM compared with parental CM. Cells treated with OxR CM demonstrated early phosphorylation of EGFR and MEK1, with later upregulation of total Akt .We identified progranulin as a potential mediator of chemoresistance.Conclusion:Chemoresistant tumour cells and CSCs may promote resistance through soluble factors that mediate survival in otherwise chemosensitive tumour cells.

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