Sensitization of Resistant Cells with a BET Bromodomain Inhibitor in a Cell Culture Model of Deep Intrinsic Resistance in Breast Cancer.

We treated highly metabolically adaptable (SUM149-MA) triple-negative inflammatory breast cancer cells and their control parental SUM149-Luc cell line with JQ1 for long periods to determine its efficacy at inhibiting therapy-resistant cells. After 20 days of treatment with 1-2 µM of JQ1, which killed majority of cells in the parental cell line, a large number of SUM149-MA cells survived, consistent with their pan-resistant nature. Interestingly, though, the JQ1 treatment sensitized resistant cancer cells in both the SUM149-MA and SUM149-Luc cell lines to subsequent treatment with doxorubicin and paclitaxel. To measure JQ1-mediated sensitization of resistant cancer cells, we first eradicated approximately 99% of relatively chemotherapy-sensitive cancer cells in culture dishes by long treatments with doxorubicin or paclitaxel, and then analyzed the remaining resistant cells for survival and growth into colonies. In addition, combination, rather than sequential, treatment with JQ1 and doxorubicin was also effective in overcoming resistance. Notably, Western blotting showed that JQ1-treated cancer cells had significantly lower levels of PD-L1 protein than did untreated cells, indicating that JQ1 treatment may reduce tumor-mediated immune suppression and improve the response to immunotherapy targeting PD-L1. Finally, JQ1 treatment with a low 62.5 nM dose sensitized another resistant cell line, FC-IBC02-MA, to treatment with doxorubicin and paclitaxel.

Evaluation of 6-mercaptopurine in a cell culture model of adaptable triple-negative breast cancer with metastatic potential.

Progenitor-like cancer cells that can survive in reversible quiescence when faced with various challenges in the body are often behind disease progression. A lack of glutamine in culture medium, which eliminates >99.9% of proliferating SUM149 triple-negative breast cancer cells, selects such adaptable, pan-resistant cells. Our data support the hypothesis that a lack of glutamine forces the selection of an epigenetic state that does not require a high level of TET2, thus selecting an "undifferentiated" therapy-resistant phenotype as seen in TET2-mutant cancers. Our data suggesting that highly adaptable cells are generated through reprograming of the epigenome and transcriptome led us to evaluate low-dose 6-mercaptopurine as a potential therapy in our model. We found that a long treatment with low-dose 6-mercaptopurine inhibited the proliferation of these adaptable cells to a greater extent than it inhibited parental cells. Importantly, a small percentage of adaptable cells survived a low-dose 6-mercaptopurine treatment in a reversible quiescence, analogous to the persistence of abnormal progenitor-like cells in inflammatory bowel disease, which stays in a durable remission with a 6-mercaptopurine treatment. Based on a biomarkers analysis, a long treatment with 6-mercaptopurine or aspirin partially reversed epithelial to mesenchymal transition in adaptable cancer cells. A cell culture model of adaptable cancer cells that persist in the body will help in discovering superior therapies that can be offered before the disease advances to metastasis.

Important Role of FTO in the Survival of Rare Panresistant Triple-Negative Inflammatory Breast Cancer Cells Facing a Severe Metabolic Challenge.

We have previously shown that only 0.01% cells survive a metabolic challenge involving lack of glutamine in culture medium of SUM149 triple-negative Inflammatory Breast Cancer cell line. These cells, designated as SUM149-MA for metabolic adaptability, are resistant to chemotherapeutic drugs, and they efficiently metastasize to multiple organs in nude mice. We hypothesized that obesity-related molecular networks, which normally help in cellular and organismal survival under metabolic challenges, may help in the survival of MA cells. The fat mass and obesity-associated protein FTO is overexpressed in MA cells. Obesity-associated cis-acting elements in non-coding region of FTO regulate the expression of IRX3 gene, thus activating obesity networks. Here we found that IRX3 protein is significantly overexpressed in MA cells (5 to 6-fold) as compared to the parental SUM149 cell line, supporting our hypothesis. We also obtained evidence that additional key regulators of energy balance such as ARID5B, IRX5, and CUX1 P200 repressor could potentially help progenitor-like TNBC cells survive in glutamine-free medium. MO-I-500, a pharmacological inhibitor of FTO, significantly (>90%) inhibited survival and/or colony formation of SUM149-MA cells as compared to untreated cells or those treated with a control compound MO-I-100. Curiously, MO-I-500 treatment also led to decreased levels of FTO and IRX3 proteins in the SUM149 cells initially surviving in glutamine-free medium as compared to MO-I-100 treatment. Interestingly, MO-I-500 treatment had a relatively little effect on cell growth of either the SUM149 or SUM149-MA cell line when added to a complete medium containing glutamine that does not pose a metabolic challenge. Importantly, once selected and cultured in glutamine-free medium, SUM149-MA cells were no longer affected by MO-I-500 even in Gln-free medium. We conclude that panresistant MA cells contain interconnected molecular networks that govern developmental status and energy balance, and genetic and epigenetic alterations that are selected during cancer evolution.