Sensitivity of Colorectal Cancer to Arginine Deprivation Therapy is Shaped by Differential Expression of Urea Cycle Enzymes

We thank Polaris Pharmaceuticals and Bio-Cancer Treatment for providing drugs and reagents. This work was supported by the Cancer Prevention Research Trust, with assistance from the Wellcome Trust Institutional Strategic Support Fund [097828/z/11/B], and Cancer Research UK in conjunction with the Department of Health as part of an Experimental Cancer Medicine Centre grant [C325/A15575]. C.A. was funded by a PhD fellowship from the Cancer Prevention Research Trust, S.S.A. was funded by a studentship from the Iraqi Government. We are thankful to John Bomalaski and Sara Galavotti for their critical reading of the manuscript and insightful suggestions. Finally, we are profoundly indebted to Professor Andreas Gescher for his constant support during the execution of this project and the writing of this manuscript.Peer reviewedPublisher PD

Assessing efficacy and molecular mechanisms of curcumin in targeting cancer stem-like cells in colorectal cancer

Curcumin inhibits the proliferation of chemotherapy-resistant cancer stem-like cells in cell lines but whether this contributes to its chemopreventive activity is unknown. This study aims to determine whether curcumin modulates the growth and expansion of colorectal stem-like cells in primary adenoma and carcinoma tissues, and in vivo using a patient-derived xenograft, then to elucidate a possible mechanism of action. Colorectal tissue obtained post-operatively (normal n=32, adenoma n=6, carcinoma n=40) was FACS profiled for markers of stem-like cells, aldehyde dehydrogenase (ALDH) activity and CD133 expression. The percentage of cells with ALDH[superscript high] activity was 11.8±1.8, 4.6±0.7 and 2.8±0.4 in adenoma, normal and carcinoma tissues, respectively. Equivalent values for CD133 expression were 1.2±0.6, 0.5±0.2 and 7.7±1.8%. To assess in vitro activity, single cells from adenomas and carcinomas (three patients each, in triplicate) were cultured as spheroids with clinically achievable curcumin concentrations. Curcumin significantly reduced adenoma and carcinoma sphere number, compared to controls for all patient samples, with a U-shaped dose-response in >50% of cases. Sphere size was also impaired at concentrations >1μM. Curcumin (0.2%) consumption in NOD/SCID mice injected (s.c) with cancer stem-like cells was associated with significant delay in time to palpable tumours, increased survival and reduced rate of tumour growth. There was also a ~60% reduction in proportion of ALDH[superscript high] cells in tumours from curcumin treated mice compared to controls (p<0.05). Curcumin (0.1, 1μM) treatment of Caco2 cells caused a significant decrease (p<0.01) in Nanog expression, an embryonic stem cell transcription factor, in cancer stem-like cells specifically. A protein pull-down assay confirmed the interaction between curcumin and Nanog. Curcumin reduced (p<0.01) Nanog phosphorylation in cancer stem-like cells which may destabilise the protein, leading to reduced levels. These results indicate that clinically achievable concentrations of curcumin target stem-like cells in colorectal adenomas and carcinomas, which may contribute to anti-cancer efficacy in humans

Inhibition of DNA-PK with AZD7648 Sensitizes Tumor Cells to Radiotherapy and Induces Type I IFN-Dependent Durable Tumor Control

PURPOSE: Combining radiotherapy (RT) with DNA damage response inhibitors may lead to increased tumor cell death through radiosensitization. DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break repair via the nonhomologous end joining (NHEJ) pathway. We hypothesized that in addition to a radiosensitizing effect from the combination of RT with AZD7648, a potent and specific inhibitor of DNA-PK, combination therapy may also lead to modulation of an anticancer immune response. EXPERIMENTAL DESIGN: AZD7648 and RT efficacy, as monotherapy and in combination, was investigated in fully immunocompetent mice in MC38, CT26, and B16-F10 models. Immunologic consequences were analyzed by gene expression and flow-cytometric analysis. RESULTS: AZD7648, when delivered in combination with RT, induced complete tumor regressions in a significant proportion of mice. The antitumor efficacy was dependent on the presence of CD8(+) T cells but independent of NK cells. Analysis of the tumor microenvironment revealed a reduction in T-cell PD-1 expression, increased NK-cell granzyme B expression, and elevated type I IFN signaling in mice treated with the combination when compared with RT treatment alone. Blocking of the type I IFN receptor in vivo also demonstrated a critical role for type I IFN in tumor growth control following combined therapy. Finally, this combination was able to generate tumor antigen-specific immunologic memory capable of suppressing tumor growth following rechallenge. CONCLUSIONS: Blocking the NHEJ DNA repair pathway with AZD7648 in combination with RT leads to durable immune-mediated tumor control

<i>Ex vivo</i> explant model of adenoma and colorectal cancer to explore mechanisms of action and patient response to cancer prevention therapies

Colorectal cancer (CRC) is the second leading cause of cancer death in the UK. Novel therapeutic prevention strategies to inhibit the development and progression of CRC would be invaluable. Potential contenders include low toxicity agents such as dietary-derived agents or repurposed drugs. However, in vitro and in vivo models used in drug development often do not take into account the heterogeneity of tumours or the tumour microenvironment. This limits translation to a clinical setting. Our objectives were to develop an ex vivo method utilizing CRC and adenoma patient-derived explants (PDEs) which facilitates screening of drugs, assessment of toxicity, and efficacy. Our aims were to use a multiplexed immunofluorescence approach to demonstrate the viability of colorectal tissue PDEs, and the ability to assess immune cell composition and interactions. Using clinically achievable concentrations of curcumin, we show a correlation between curcumin-induced tumour and stromal apoptosis (P < .001) in adenomas and cancers; higher stromal content is associated with poorer outcomes. B cell (CD20+ve) and T cell (CD3+ve) density of immune cells within tumour regions in control samples correlated with curcumin-induced tumour apoptosis (P < .001 and P < .05, respectively), suggesting curcumin-induced apoptosis is potentially predicted by baseline measures of immune cells. A decrease in distance between T cells (CD3+ve) and cytokeratin+ve cells was observed, indicating movement of T cells (CD3+ve) towards the tumour margin (P < .001); this change is consistent with an immune environment associated with improved outcomes. Concurrently, an increase in distance between T cells (CD3+ve) and B cells (CD20+ve) was detected following curcumin treatment (P < .001), which may result in a less immunosuppressive tumour milieu. The colorectal tissue PDE model offers significant potential for simultaneously assessing multiple biomarkers in response to drug exposure allowing a greater understanding of mechanisms of action and efficacy in relevant target tissues, that maintain both their structural integrity and immune cell compartments.</p

Sulfate metabolites provide an intracellular pool for resveratrol generation and induce autophagy with senescence

The phytochemical resveratrol has been shown to exert numerous health benefits in preclinical studies, but its rapid metabolism and resulting poor bioavailability may limit translation of these effects to humans. Resveratrol metabolites might contribute to in vivo activity through regeneration of the parent compound. We present quantitation of sulfate and glucuronide conjugates of resveratrol in human plasma and tissue after repeated ingestion of resveratrol by volunteers and cancer patients, respectively. Subsequent pharmacokinetic characterization of a mixture of resveratrol-3-O-sulfate and resveratrol-4′-O-sulfate in mice showed that these metabolites are absorbed orally but have low bioavailabilities of ~14 and 3%, respectively. Sulfate hydrolysis in vivo liberated free resveratrol, which accounted for ~2% of the total resveratrol species present in mouse plasma. Monosulfate metabolites were also converted to the parent in human colorectal cells. The extent of cellular uptake was dependent on specific membrane transporters and dictated antiproliferative activity. Sulfate metabolites induced autophagy and senescence in human cancer cells; these effects were abrogated by inclusion of a sulfatase inhibitor, which reduced intracellular resveratrol. Together, our findings suggest that resveratrol is delivered to target tissues in a stable sulfate-conjugated form and that the parent compound is gradually regenerated in selected cells and may give rise to the beneficial effects in vivo. At doses considered to be safe in humans, resveratrol generated via this route may be of greater importance than the unmetabolized form

Flow cytometric profiling of spheroids under co-culture conditions.

<p>(A) Original sample profiles expressed as a percent of total cells, and as a percent of EpCAM⁺ cells. (B) Samples following one passage with 18Co cells expressed as a percent of total cells, and as a percent of EpCAM⁺ cells.</p

Comparison of TIC marker expression between an original tissue sample, matched spheroids and differentiated cells.

<p>(A) comparison of TIC marker expression between tissue, spheroids and differentiated cultures from the same patient (N = 1 for tissue (stats not shown), N = 4 for spheroids and N = 3 for differentiated cells, mean ± SEM, paired sample t-test, * = P≤0.05). (B) TIC marker expression comparing original spheroids with spheroids produced from differentiated cells when placed under low adherence conditions (N = 3, mean ± SEM, paired sample t-test, * = P≤0.05).</p

Flow cytometry data (EpCAM^<b>+</b> cells only) showing TIC populations by gender and chemotherapy status.

<p>Flow cytometry data (EpCAM^<b>+</b> cells only) showing TIC populations by gender and chemotherapy status.</p

Patient demographics.

<p>Patient demographics.</p

TIC marker expression in colorectal liver metastases.

<p>(A) Shows mean (±SEM) expression of TIC markers analysed by flow cytometry from patients undergoing resection for CRLM. N = 62 for ALDH combinations, N = 44 for CD26 combinations. (B) Mean (±SEM) TIC marker expression comparing males vs females. Male N = 39 and 26 for ALDH and CD26 combinations respectively, female N = 22 and 17 for ALDH and CD26 combinations respectively (independent t-tests, * = P≤0.05). (C) TIC marker expression in EpCAM⁺ cells stratified based on patient chemotherapy status. Chemo naïve N = 14, chemo-treated N = 36. (mean ± SEM, Mann Whitney-U test, * indicates P = 0.05). (D) Representative dot plot for CD26 (x-axis) and CD133 (y-axis) in a chemo-naïve (left) and chemo-treated (right) patient.</p