Investigating the role of the stress-related transcription factor, HSF1, upon breast cancer tumourigenesis and progression
2017-02-28T04:19:23Z (GMT) by
Cancer cells are exposed to numerous forms of extrinsic and intrinsic cellular stresses such as hypoxia, acidosis, nutrient deprivation, as well as genotoxic and oxidative stress, and as a result are dependent upon stress support pathways for their survival. One such pathway is the heat shock response (HSR), which results in the enhanced expression of heat shock proteins (HSPs) that function as molecular chaperones that restore cellular protein homeostasis and prevent stress-induced cell death. However, cellular stress has also been shown to be an important contributor to cancer cell growth, progression and metastasis. The elevated expression of HSPs has been identified in many tumour types and correlates with poor patient outcomes. As such, HSPs have emerged as significant therapeutic targets within many cancer types. Consistent with this, the master transcription factor regulating the HSR, Heat Shock Factor 1 (HSF1), has also been shown to function as a powerful modulator of malignancy. Interestingly, HSF1 is found to not only support the malignant phenotype by regulating the expression of the HSPs but also regulates the expression of a complex network of genes that are involved in many cellular processes essential for tumourigenesis and cancer progression. However, while there have been many studies that document the important roles of HSF1 in cancer; the precise mechanisms by which HSF1 achieves this are still relatively unknown. Despite significant improvements over the years in cancer treatment, breast cancer remains a major cause of death among women worldwide. In particular, individuals diagnosed with triple negative forms of breast cancer that are more refractory to current therapies and have a higher likelihood to undergo metastasis, have particularily poor outcomes. Thus, the identification of novel and more effective therapeutic drug targets to improve patient survival is required. Previous studies have revealed that elevated levels and increased activity of HSF1 are strongly correlated with breast cancer aggressiveness and outcome. Analysis of breast cancer cell lines has also demonstrated that HSF1 levels and activity are increased in highly aggressive and metastatic triple negative cancer cell lines in comparison to the lower migratory and less invasive luminal breast cancer cell line subtypes. As HSF1 has emerged as a potential anticancer therapeutic target, this study aims to validate and determine the mechanisms by which HSF1 may act in breast cancer tumourigenesis and progression. In this study, to investigate the role of HSF1 in breast cancer tumourigenesis and progression, wild-type HSF1 and a constitutively active form of HSF1, HSF1ΔRDT, were ectopically expressed in the normal human mammary epithelial cell line, MCF10A, and in MCF10A H-RasV12 transformed cells. This study demonstrates that while ectopic expression of HSF1 has little impact upon the cell biology of the normal MCF10A cells, HSF1 uniquely enhances the malignant phenotype of cells that have been transformed with oncogenic Ras, especially in regard to the cells’ migratory and invasion abilities. Similar effects were observed when HSF1 was ectopically expressed in the luminal breast cancer cell line, SkBr3, which exhibits a constitutive activation of Ras. Further analysis reveals that while HSF1 exerts little effects on signal transduction pathways downstream of Ras, the factor co-operates with oncogenic Ras to alter the expression of genes and pathways that promote cancer progression. This study thus confirms that HSF1 is a positive modulator of cancer progression and shows that the cancer promoting effects of HSF1 are mediated via the modulation and/or co-operation of the factor with other oncogenic proteins within the tumour cells. In addition to its co-operative actions with activated oncogenic Ras, this study also demonstrates that HSF1 can regulate breast cancer cell clonogenicity and this activity is dependent upon the tumour suppressor p53. Wild-type p53 functions as a “guardian of the genome” that regulates the expression of genes involved in DNA damage repair, cell-cycle arrest and apoptosis. Mutations in the TP53 gene lead to the production of mutant p53 proteins that not only exhibit a loss in their tumour suppressor activity but can also exert ‘gain-of-function’ properties that have been shown to be important at key stages of metastatic progression. This study demonstrates that HSF1 can enhance both wild-type and mutant p53 transcriptional activities, mediating disparate outcomes in clonogenic cancer cell survival and growth in a p53 status dependent manner. Knockdown of mutant p53 abrogates HSF1’s ability to enhance clonogenic survival and growth in cancer cells, while knockdown of wild-type p53 rescues the reduced clonogenicity that is mediated by HSF1 ectopic expression. Moreover, in the cellular context of endogenous wild-type p53 and the exogenous expression of mutant p53R273H, activation of HSF1 reduces cell clonogenicity; however, when wild-type p53 is knocked down leaving a cellular context of mutant p53R273H, activation of HSF1 can support p53R273H activities, thereby greatly increasing clonogenic survival and growth. Therefore, these findings demonstrate that HSF1 actions can be cell context dependent with respect to p53 status. In addition, this study has also generated HSF1 shRNAmir constructs and examined the effects of HSF1 knockdown within differing cellular contexts. While previous studies have demonstrated that inhibition of HSF1 can abrogate the malignant phenotype of many high-grade cancer cells, this study demonstrates that inhibition of HSF1 exerts little impact upon the cell biology of normal and H-RasV12 transformed MCF10A cells. However, HSF1 knockdown reduces the clonogenicity of these cells, not only by the reduction of HSP expression, but also potentially through increasing the steady state levels and activity of wild-type p53. Together with previous studies, this work indicates that the inhibition of HSF1 would uniquely abrogate the growth of high-grade tumours while exerting minimal toxicity to normal cells. Moreover, HSF1 inhibition could potentially be used to enhance the efficacy of cancer therapies that activate wild-type p53. Finally, while there is currently a lack of specific and/or potent HSF1 inhibitors, this study has also successfully developed a novel cell-based reporter system that could be used for large-scale HSF1 inhibitor screening. The development of this model could lead to the identification of new therapeutic compounds for anticancer treatment. In summary, these studies support the notion that HSF1 is not an oncogene per se but rather functions as an enhancer of cancer progression by supporting the maintenance of malignant phenotypes induced by other genetic and epigenetic alterations within tumour cells. In particular, this study shows that HSF1 exerts disparate effects upon cancer tumourigenesis and progression with respect to differing cellular oncogenic contexts. Therefore this work adds to our understanding of the role of HSF1 in cancer cell survival and progression and has important implications for its therapeutic targeting in cancer treatments.