Epigenetic reprogramming and potential application of epigenetic-modifying drugs in acquired chemotherapeutic resistance.

Chemotherapy is the most common clinical choice of treatment for cancer, however, acquired chemoresistance is a major challenge that limits the successful outcome of this option. Systematic review of in vitro, in vivo, preclinical and clinical studies suggests that acquired chemoresistance is polygenic, progressive, and involve both genetic and epigenetic heterogeneities and perturbations. Various mechanisms that confer resistance to chemotherapy are tightly controlled by epigenetic regulations. Poised epigenetic plasticity and temporal increase in epigenetic alterations upon chemotherapy make chemoresistance likely an epigenetic-driven process. The transient and reversible nature of epigenetic modulations enable ways to intervene the epigenetic re-programing associated with acquired chemoresistance via application of epigenetic modifying drugs. This review discusses recent understandings behind the various mechanisms of acquired chemoresistance that are under the control of epigenetic drivers, potential application of epigenetic-based drugs in resensitizing refractory cancers to chemotherapy, the limitations and future scope for clinical application of epigenetic therapeutics in successfully addressing chemoresistance.

Role of cellular reprogramming and epigenetic dysregulation in acquired chemoresistance in breast cancer.

Acquired resistance to chemotherapy is a major limitation in clinical treatment for breast cancer. Accumulating evidence from in vitro, in vivo and clinical studies suggest that acquired chemoresistance is progressive, multifactorial and involve genetic and epigenetic aberrations. Among various mechanisms that contribute to chemoresistance, cellular reprogramming has extensively been implicated in breast cancer resistance lately. Cellular reprogramming events such as acquisition of epithelial to mesenchymal transition (EMT) and cancer stemness (CSCs) not only provide cancer cells with reversible phenotypic plasticity and survival advantage against cytotoxicity but also leads to aggressiveness, metastasis, clinical resistance, tumor recurrence and poor survival. The transient and reversible nature of cellular reprogramming processes and their controlled interaction with epigenetic regulatory complexes strongly support the involvement of dynamic epigenetic regulatory network in governing the cellular reprogramming and associated acquired chemoresistance. Further, epigenetic modulations are also gaining interest as promising interventions addressing the cancer cell reprogramming machinery to overcome acquired chemoresistance. This review discusses the previous reports and our recent findings that lead to current understanding of epigenetic dysregulation dictating the cellular reprogramming processes such as acquisition of EMT and CSCs phenotype and how they co-ordinate to establish acquired drug resistance in breast cancer.

Reversal of epigenetic aberrations associated with the acquisition of doxorubicin resistance restores drug sensitivity in breast cancer cells.

Acquired resistance against doxorubicin is a major limitation in clinical treatment of breast cancer. The molecular mechanism behind the aberrant expression of genes leading to doxorubicin resistance is not clear. Epigenetic changes play an important role in the regulation of gene expression. Therefore, the objective of this study was to identify the epigenetic mechanism underlying acquired doxorubicin resistance in breast cancer cells. Doxorubicin-resistant cells were selected by repeated exposure of MCF-7 and MDA-MB-231 breast cancer cell lines to clinically relevant doses of doxorubicin for 18 months. MTT assay, cell cycle analysis, colony formation, qRT-PCR, and Western blot analyses were used to characterize the epigenetic and molecular mechanism. Pyrosequencing was used to detect MSH2 promoter hypermethylation. Aberrant expression of epigenetic regulatory genes, a significant increase in H3 acetylation and methylation, as well as promoter hypermethylation-mediated inactivation of MSH2 gene were associated with the acquired resistant phenotype. Demethylating agent 5-Aza-deoxycytidine and HDAC inhibitor Trichostatin A significantly re-sensitized resistant cells to doxorubicin. Findings of this study revealed that epigenetic aberrations including promoter hypermethylation-mediated inactivation MSH2 contribute to the acquisition of doxorubicin resistance in breast cancer cells. Additionally, our data suggest that some of these epigenetic aberrations are progressive during resistance development and therefore can potentially be used as biomarkers for early detection of resistance. These epigenetic aberrations, being reversible, can also serve as targets for epigenetic therapy to re-sensitize doxorubicin-resistant breast cancer cells. Epigenetic inactivation of mismatch repair gene MSH2 further suggests that loss of MMR-dependent apoptotic potential could be a novel mechanistic basis for the acquisition of doxorubicin resistance in breast cancer cells.

Treatment schedule and estrogen receptor-status influence acquisition of doxorubicin resistance in breast cancer cells.

Breast cancer is the most common cancer in women for which doxorubicin is still the mainstay treatment. However, chemotherapy resistance is a major limitation in breast cancer treatment. Role of treatment schedule and estrogen receptor (ER) status in subtypes of breast cancers in acquired resistance development is not clear. Therefore, objective of this study was to evaluate whether the treatment schedule and ER status in breast cancer cells influence the doxorubicin resistance. To address these questions, ER-positive MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines were given either continuous or intermittent exposure with clinically relevant concentration of doxorubicin and the influence of these two treatment strategies on resistance to drug sensitivity was evaluated. Results revealed that intermittent treatment but not the continuous treatment induced resistance in breast cancer cells against doxorubicin. MCF-7 cells developed relatively earlier and high level of resistance when compared to MDA-MB-231 cells. Acquisition of epithelial to mesenchymal transition (EMT) and cancer stem cell-like phenotype was also observed during resistance development in MCF-7 cells. Changes in the expression of selected marker genes including drug transporters confirmed doxorubicin resistance in these cells. In summary, this study suggests that acquisition of resistance against doxorubicin depends on the treatment schedule of this drug as well as the estrogen receptor-based subtypes of breast cancer. Additionally, acquisition of EMT and stem cell-like phenotype further provided a molecular basis for breast cancer subtype-dependent chemotherapeutic resistance development. Findings of this study will have significant clinical implications in optimizing the chemotherapy schedule to minimize chemoresistance in breast cancer patients.

Oxidative stress-induced epigenetic changes associated with malignant transformation of human kidney epithelial cells.

Renal Cell Carcinoma (RCC) in humans is positively influenced by oxidative stress status in kidneys. We recently reported that adaptive response to low level of chronic oxidative stress induces malignant transformation of immortalized human renal tubular epithelial cells. Epigenetic alterations in human RCC are well documented, but its role in oxidative stress-induced malignant transformation of kidney cells is not known. Therefore, the objective of this study was to evaluate the potential role of epigenetic changes in chronic oxidative stress-induced malignant transformation of HK-2, human renal tubular epithelial cells. The results revealed aberrant expression of epigenetic regulatory genes involved in DNA methylation (DNMT1, DNMT3a and MBD4) and histone modifications (HDAC1, HMT1 and HAT1) in HK-2 cells malignantly transformed by chronic oxidative stress. Additionally, both in vitro soft agar assay and in vivo nude mice study showing decreased tumorigenic potential of malignantly transformed HK-2 cells following treatment with DNA de-methylating agent 5-aza 2' dC further confirmed the crucial role of DNA hypermethyaltion in oxidative stress-induced malignant transformation. Changes observed in global histone H3 acetylation (H3K9, H3K18, H3K27 and H3K14) and decrease in phospho-H2AX (Ser139) also suggest potential role of histone modifications in increased survival and malignant transformation of HK-2 cells by oxidative stress. In summary, the results of this study suggest that epigenetic reprogramming induced by low levels of oxidative stress act as driver for malignant transformation of kidney epithelial cells. Findings of this study are highly relevant in potential clinical application of epigenetic-based therapeutics for treatments of kidney cancers.

Chronic Oxidative Stress Increases Resistance to Doxorubicin-Induced Cytotoxicity in Renal Carcinoma Cells Potentially Through Epigenetic Mechanism.

Renal cell carcinoma is the most common form of kidney cancer and is highly resistant to chemotherapy. Although the role of oxidative stress in kidney cancer is known, the chemotherapeutic response of cancer cells adapted to chronic oxidative stress is not clear. Hence, the effect of oxidative stress on sensitivity to doxorubicin-induced cytotoxicity was evaluated using an in vitro model of human kidney cancer cells adapted to chronic oxidative stress. Results of MTT- and anchorage-independent growth assays and cell cycle analysis revealed significant decrease in sensitivity to doxorubicin in Caki-1 cells adapted to oxidative stress. Changes in the expression of genes involved in drug transport, cell survival, and DNA repair-dependent apoptosis further confirmed increased resistance to doxorubicin-induced cytotoxicity in these cells. Decreased expression of mismatch repair (MMR) gene MSH2 in cells exposed to oxidative stress suggests that loss of MMR-dependent apoptosis could be a potential mechanism for increased resistance to doxorubicin-induced cytotoxicity. Additionally, downregulation of HDAC1, an increase in the level of histone H3 acetylation, and hypermethylation of MSH2 promoter were also observed in Caki-1 cells adapted to chronic oxidative stress. DNA-demethylating agent 5-Aza-2dC significantly restored the expression of MSH2 and doxorubicin-induced cytotoxicity in Caki-1 cells adapted to chronic oxidative stress, suggesting the role of DNA hypermethylation in inactivation of MSH2 expression and consequently MMR-dependent apoptosis in these cells. In summary, this study for the first time provides direct evidence for the role of oxidative stress in chemotherapeutic resistance in renal carcinoma cells potentially through epigenetic mechanism.

Chronic oxidative stress causes estrogen-independent aggressive phenotype, and epigenetic inactivation of estrogen receptor alpha in MCF-7 breast cancer cells.

The role of chronic oxidative stress in the development and aggressive growth of estrogen receptor (ER)-positive breast cancer is well known; however, the mechanistic understanding is not clear. Estrogen-independent growth is one of the features of aggressive subtype of breast cancer. Therefore, the objective of this study was to evaluate the effect of oxidative stress on estrogen sensitivity and expression of nuclear estrogen receptors in ER-positive breast cancer cells. MCF-7 cells chronically exposed to hydrogen peroxide were used as a cell model in this study, and their growth in response to 17-ß estradiol was evaluated by cell viability, cell cycle, and cell migration analysis. Results were further confirmed at molecular level by analysis of gene expressions at transcript and protein levels. Histone H3 modifications, expression of epigenetic regulatory genes, and the effect of DNA demethylation were also analyzed. Loss of growth in response to estrogen with a decrease in ERα expression was observed in MCF-7 cells adapted to chronic oxidative stress. Increases in mtTFA and NRF1 in these cells further suggested the role of mitochondria-dependent redox-sensitive growth signaling as an alternative pathway to estrogen-dependent growth. Changes in expression of epigenetic regulatory genes, levels of histone H3 modifications as well as significant restorations of both ERα expression and estrogen response by 5-Aza-2'-deoxycytidine further confirmed the epigenetic basis for estrogen-independent growth in these cells. In conclusion, results of this study suggest that chronic oxidative stress can convert estrogen-dependent nonaggressive breast cancer cells into estrogen-independent aggressive form potentially by epigenetic mechanism.

Chronic oxidative stress leads to malignant transformation along with acquisition of stem cell characteristics, and epithelial to mesenchymal transition in human renal epithelial cells.

Oxidative injury to cellular macromolecules has been suggested as a common pathway shared by multiple etiological factors for kidney cancer. Whether the chronic oxidative stress alone is sufficient to induce malignant transformation in human kidney cells is not clear. Therefore, the objective of this study was to evaluate the effect of H2O2-induced chronic oxidative stress on growth, and malignant transformation of HK-2 normal kidney epithelial cells. This study revealed that chronic oxidative stress causes increased growth and neoplastic transformation in normal kidney epithelial cells at non-cytotoxic dose and increased adaptation to cytotoxic level. This was confirmed by gene expression changes, cell cycle analysis, anchorage independent growth assay and in vivo tumorigenicity in nude mice. Stem cells characteristics as revealed by up-regulation of stem cell marker genes, and morphological changes indicative of EMT with up regulation of mesenchymal markers were also observed in cells exposed to chronic oxidative stress. Antioxidant NAC did not reverse the chronic oxidative stress-induced growth, and adaptation suggesting that perturbed biological function in these cells are permanent. Partial reversal of oxidative stress-induced growth, and adaptation by silencing of Oct 4 and Snail1, respectively, suggest that these changes are mediated by acquisition of stem cell and EMT characteristics. In summary, this study for the first time suggests that chronic exposure to elevated levels of oxidative stress is sufficient to induce malignant transformation in kidney epithelial cells through acquisition of stem cell characteristics. Additionally, the EMT plays an important role in increased adaptive response of renal cells to oxidative stress.