These fundamental observations may assist physicians in evaluating the survival potential of patients and in directing them toward the appropriate therapeutic decision.

The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional mode of hypoxia-inducible factor (HIF)-dependent transcriptional regulation, involving modulation of a specific set of micro RNAs (miRNAs), including miR-210, has emerged. We have recently shown that HIF-1 induction of miR-210 also stabilizes HIF-1 through a positive regulatory loop. Therefore, we hypothesized that by stabilizing HIF-1 in normoxia, miR-210 may protect cancer cells from radiation. We developed a non-small cell lung carcinoma (NSCLC)-derived cell line (A549) stably expressing miR-210 (pmiR-210) or a control miRNA (pmiR-Ctl). The miR-210-expressing cells showed a significant stabilization of HIF-1 associated with mitochondrial defects and a glycolytic phenotype. Cells were subjected to radiation levels ranging from 0 to 10 Gy in normoxia and hypoxia. Cells expressing miR-210 in normoxia had the same level of radioresistance as control cells in hypoxia. Under hypoxia, pmiR-210 cells showed a low mortality rate owing to a decrease in apoptosis, with an ability to grow even at 10 Gy. This miR-210 phenotype was reproduced in another NSCLC cell line (H1975) and in HeLa cells. We have established that radioresistance was independent of p53 and cell cycle status. In addition, we have shown that genomic double-strand breaks (DSBs) foci disappear faster in pmiR-210 than in pmiR-Ctl cells, suggesting that miR-210 expression promotes a more efficient DSB repair. Finally, HIF-1 invalidation in pmiR-210 cells removed the radioresistant phenotype, showing that this mechanism is dependent on HIF-1. In conclusion, miR-210 appears to be a component of the radioresistance of hypoxic cancer cells. Given the high stability of most miRNAs, this advantage could be used by tumor cells in conditions where reoxygenation has occurred and suggests that strategies targeting miR-210 could enhance tumor radiosensitization.

Resistance to chemotherapy-induced apoptosis of tumor cells represents a major hurdle to efficient cancer therapy. Although resistance is a characteristic of tumor cells that evolve in a low oxygen environment (hypoxia), the mechanisms involved remain elusive. We observed that mitochondria of certain hypoxic cells take on an enlarged appearance with reorganized cristae. In these cells, we found that a major mitochondrial protein regulating metabolism and apoptosis, the voltage-dependent anion channel 1 (VDAC1), was linked to chemoresistance when in a truncated (VDAC1-DC) but active form. The formation of truncated VDAC1, which had a similar channel activity and voltage dependency as full-length, was hypoxia-inducible factor-1 (HIF-1)-dependent and could be inhibited in the presence of the tetracycline antibiotics doxycycline and minocycline, known inhibitors of metalloproteases. Its formation was also reversible upon cell reoxygenation and associated with cell survival through binding to the antiapoptotic protein hexokinase. Hypoxic cells containing VDAC1-DC were less sensitive to staurosporine-and etoposide-induced cell death, and silencing of VDAC1-DC or treatment with the tetracycline antibiotics restored sensitivity. Clinically, VDAC1-DC was detected in tumor tissues of patients with lung adenocarcinomas and was found more frequently in large and late-stage tumors. Together, our findings show that via induction of VDAC1-DC, HIF-1 confers selective protection from apoptosis that allows maintenance of ATP and cell survival in hypoxia. VDAC1-DC may also hold promise as a biomarker for tumor progression in chemotherapy-resistant patients. Cancer Res; 72(8); 2140-50. Ó2012 AACR.

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The relationship between acidosis within the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases (CA) IX and CAXII constitute a robust intracellular pH (pHi)-regulating system that confers a survival advantage on hypoxic human colon carcinoma LS174Tr cells in acidic microenvironments. Here we investigate the role of acidosis, CAIX and CAXII knock-down in combination with ionizing radiation. Fibroblasts cells (-/+ CAIX) and LS174Tr cells (inducible knock-down for ca9/ca12) were analyzed for cell cycle phase distribution and survival after irradiation in extracellular pHo manipulations and hypoxia (1% O2) exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pHi-regulating capacity of fibroblasts through inhibition of Na+/H+ exchanger 1 sensitize cells to radiation-induced cell death. Secondly, the pHi-regulating function of CAIX plays a key protective role in irradiated fibroblasts in an acidic environment as accompanied by a reduced number of cells in the radiosensitive phases of the cell cycle. Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50 and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pHi regulation. Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo. These findings highlight the combinatory use of radiotherapy with targeting of the pHi-regulating CAs as an anti-cancer strategy.

Purpose: No biomarker to personalize treatment in locally advanced rectal cancer (LARC) is currently available. We assessed in LARC whether a diagnostic biopsy-adapted immunoscore (IS B) could predict response to neoadjuvant treatment (nT) and better define patients eligible to an organ preservation strategy ("Watchand-Wait"). Experimental Design: Biopsies from two independent cohorts (n 1 ¼ 131, n 2 ¼ 118) of patients with LARC treated with nT followed by radical surgery were immunostained for CD3 þ and CD8 þ T cells and quantified by digital pathology to determine IS B. The expression of immune-related genes post-nT was investigated (n ¼ 64 patients). Results were correlated with response to nT and disease-free survival (DFS). The IS B prognostic performance was further assessed in a multicentric cohort (n ¼ 73 patients) treated by Watch-and-Wait. Results: IS B positively correlated with the degree of histologic response (P < 0.001) and gene expression levels for Th1 orientation and cytotoxic immune response, post-nT (P ¼ 0.006). IS B high identified patients at lower risk of relapse or death compared with IS B low [HR, 0.21; 95% confidence interval (CI), 0.06-0.78; P ¼ 0.009]. Prognostic performance of IS B for DFS was confirmed in a validation cohort. IS B was an independent parameter, more informative than pre-(P < 0.001) and post-nT (P < 0.05) imaging to predict DFS. IS B combined with imaging post-nT discriminated very good responders that could benefit from organ preservation strategy. In the "Watch-and-Wait" cohort (n ¼ 73), no relapse was observed in patients with IS B high (23.3%). Conclusions: IS B predicts response to nT and survival in patients with LARC treated by surgery. Its usefulness in the selection of patients eligible for a Watch-and-Wait strategy is strongly suggested.

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