Lysosomal inhibition sensitizes TMEM16A-expressing cancer cells to chemotherapy.

Squamous cell carcinoma of the head and neck (SCCHN) is a devastating disease that continues to have low cure rates despite the recent advances in therapies. Cisplatin is the most used chemotherapy agent, and treatment failure is largely driven by resistance to this drug. Amplification of chromosomal band 11q13 occurs in ∼30% of SCCHN tumors. This region harbors the ANO1 gene that encodes the TMEM16A ion channel, which is responsible for calcium-activated chloride transport in epithelial tissues. TMEM16A overexpression is associated with cisplatin resistance, and high TMEM16A levels correlate with decreased survival. However, the mechanistic underpinning of this effect remains unknown. Lysosomal biogenesis and exocytosis have been implicated in cancer because of their roles in the clearance of damaged organelles and exocytosis of chemotherapeutic drugs and toxins. Here, we show that TMEM16A overexpression promotes lysosomal biogenesis and exocytosis, which is consistent with the expulsion of intracellular cisplatin. Using a combination of genetic and pharmacologic approaches, we find that TMEM16A promotes lysosomal flux in a manner that requires reactive oxygen species, TRPML1, and the activation of the ß-catenin­melanocyte-inducing transcription factor pathway. The lysosomal inhibitor hydroxychloroquine (HCQ) synergizes with cisplatin in killing SCCHN cells in vitro. Using a murine model of SCCHN, we show that HCQ and cisplatin retard the growth of cisplatin-resistant patient-derived xenografts in vivo. We propose that TMEM16A enables cell survival by the up-regulation of lysosomal sequestration and exocytosis of the cytotoxic drugs. These results uncover a model of treatment for resistance in cancer, its reversal, and a role for TMEM16A.

TMEM16A/ANO1 Inhibits Apoptosis Via Downregulation of Bim Expression.

Purpose: TMEM16A is a calcium-activated chloride channel that is amplified in a variety of cancers, including 30% of head and neck squamous cell carcinomas (HNSCCs), raising the possibility of an anti-apoptotic role in malignant cells. This study investigated this using a multimodal, translational investigation.Experimental Design: Combination of (i) in vitro HNSCC cell culture experiments assessing cell viability, apoptotic activation, and protein expression (ii) in vivo studies assessing similar outcomes, and (iii) molecular and staining analysis of human HNSCC samples.Results: TMEM16A expression was found to correlate with greater tumor size, increased Erk 1/2 activity, less Bim expression, and less apoptotic activity overall in human HNSCC. These findings were corroborated in subsequent in vitro and in vivo studies and expanded to include a cisplatin-resistant phenotype with TMEM16A overexpression. A cohort of 41 patients with laryngeal cancer demonstrated that cases that recurred after chemoradiation failure were associated with a greater TMEM16A overexpression rate than HNSCC that did not recur.Conclusions: Ultimately, this study implicates TMEM16A as a contributor to tumor progression by limiting apoptosis and as a potential biomarker of more aggressive disease. Clin Cancer Res; 23(23); 7324-32. ©2017 AACR.

Targeting cancer stem cells with p53 modulators.

Cancer stem cells (CSC) typically over-express aldehyde dehydrogenase (ALDH). Thus, ALDHbright tumor cells represent targets for developing novel cancer prevention/treatment interventions. Loss of p53 function is a common genetic event during cancer development wherein small molecular weight compounds (SMWC) that restore p53 function and reverse tumor growth have been identified. Here, we focused on two widely studied p53 SMWC, CP-31398 and PRIMA-1, to target ALDHbright CSC in human breast, endometrial and pancreas carcinoma cell lines expressing mutant or wild type (WT) p53. CP-31398 and PRIMA-1 significantly reduced CSC content and sphere formation by these cell lines in vitro. In addition, these agents were more effective in vitro against CSC compared to cisplatin and gemcitabine, two often-used chemotherapeutic agents. We also tested a combinatorial treatment in methylcholantrene (MCA)-treated mice consisting of p53 SMWC and p53-based vaccines. Yet using survival end-point analysis, no increased efficacy in the presence of either p53 SMWC alone or with vaccine compared to vaccine alone was observed. These results may be due, in part, to the presence of immune cells, such as activated lymphocytes expressing WT p53 at levels comparable to some tumor cells, wherein further increase of p53 expression by p53 SMWC may alter survival of these immune cells and negatively impact an effective immune response. Continuous exposure of mice to MCA may have also interfered with the action of these p53 SMWC, including potential direct interaction with MCA. Nonetheless, the effect of p53 SMWC on CSC and cancer treatment remains of great interest.

Radioresistant human lung adenocarcinoma cells that survived multiple fractions of ionizing radiation are sensitive to HSP90 inhibition.

Despite the common usage of radiotherapy for the treatment of NSCLC, outcomes for these cancers when treated with ionizing radiation (IR) are still unsatisfactory. A better understanding of the mechanisms underlying resistance to IR is needed to design approaches to eliminate the radioresistant cells and prevent tumor recurrence and metastases. Using multiple fractions of IR we generated radioresistant cells from T2821 and T2851 human lung adenocarcinoma cells. The radioresistant phenotypes present in T2821/R and T2851/R cells include multiple changes in DNA repair genes and proteins expression, upregulation of EMT markers, alterations of cell cycle distribution, upregulation of PI3K/AKT signaling and elevated production of growth factors, cytokines, important for lung cancer progression, such as IL-6, PDGFB and SDF-1 (CXCL12). In addition to being radioresistant these cells were also found to be resistant to cisplatin.HSP90 is a molecular chaperone involved in stabilization and function of multiple client proteins implicated in NSCLC cell survival and radioresistance. We examined the effect of ganetespib, a novel HSP90 inhibitor, on T2821/R and T2851/R cell survival, migration and radioresistance. Our data indicates that ganetespib has cytotoxic activity against parental T2821 and T2851 cells and radioresistant T2821/R and T2851/R lung tumor cells. Ganetespib does not affect proliferation of normal human lung fibroblasts. Combining IR with ganetespib completely abrogates clonogenic survival of radioresistant cells.Our data show that HSP90 inhibition can potentiate the effect of radiotherapy and eliminate radioresistant and cisplatin -resistant residual cells, thus it may aid in reducing NSCLC tumor recurrence after fractionated radiotherapy.

The HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells.

The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC) cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of ß-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors.

Non-small cell lung cancer cells survived ionizing radiation treatment display cancer stem cell and epithelial-mesenchymal transition phenotypes.

Ionizing radiation (IR) is used for patients diagnosed with unresectable non small cell lung cancer (NSCLC), however radiotherapy remains largely palliative due to radioresistance. Cancer stem cells (CSCs), as well as epithelial-mesenchymal transition (EMT), may contribute to drug and radiation resistance mechanisms in solid tumors. Here we investigated the molecular phenotype of A549 and H460 NSCLC cells that survived treatment with IR (5Gy) and are growing as floating tumor spheres and cells that are maintained in a monolayer after irradiation.Non-irradiated and irradiated cells were collected after one week, seeded onto ultra low attachment plates and propagated as tumor spheres. Bulk NSCLC cells which survived radiation and grew in spheres express cancer stem cell surface and embryonic stem cell markers and are able to self-renew, and generate differentiated progeny. These cells also have a mesenchymal phenotype. Particularly, the radiation survived sphere cells express significantly higher levels of CSC markers (CD24 and CD44), nuclear ß-catenin and EMT markers (Snail1, Vimentin, and N-cadherin) than non-irradiated lung tumor sphere cells. Upregulated levels of Oct-4, Sox2 and beta-catenin were detected in H460 cells maintained in a monolayer after irradiation, but not in radiation survived adherent A459 cells.PDGFR-beta was upregulated in radiation survived sphere cells and in radiation survived adherent cells in both A549 and H460 cell lines. Combining IR treatment with axitinib or dasatinib, inhibitors with anti-PDFGR activity, potentiates the efficacy of NSCLC radiotherapy in vitro.Our findings suggest that radiation survived cells have a complex phenotype combining the properties of CSCs and EMT. CD44, SNAIL and PDGFR-beta are dramatically upregulated in radiation survived cells and might be considered as markers of radiotherapy response in NSCLC.