SUMMARY Signaling through RAS/MAP kinase pathway is central to biology. ERK has long been perceived as the only substrate for MEK. Herein we report that HSF1, the master regulator of the proteotoxic stress response, is a new MEK substrate. Beyond mediating cell-environment interactions, the MEK-HSF1 regulation impacts malignancy. In tumor cells, MEK blockade inactivates HSF1 and thereby provokes proteomic chaos, presented as protein destabilization, aggregation, and, strikingly, amyloidogenesis. Unlike their non-transformed counterparts, tumor cells are particularly susceptible to proteomic perturbation and amyloid induction. Amyloidogenesis is tumor-suppressive, reducing in vivo melanoma growth and contributing to the potent anti-neoplastic effects of proteotoxic stressors. Our findings unveil a key biological function of the oncogenic RAS-MEK signaling in guarding proteostasis and suppressing amyloidogenesis. Thus, proteomic instability is an intrinsic feature of malignant state and, disrupting the fragile tumor proteostasis to promote amyloidogenesis may be a feasible therapeutic strategy.

Intrinsic stress response pathways are frequently mobilized within tumor cells. The mediators of these adaptive mechanisms and how they contribute to carcinogenesis remain poorly understood. A striking example is heat shock factor 1 (HSF1), master transcriptional regulator of the heat shock response. Surprisingly, we found that loss of the tumor suppressor gene neurofibromatosis type 1 (Nf1) increased HSF1 levels and triggered its activation in mouse embryonic fibroblasts. As a consequence, Nf1 -/-cells acquired tolerance to proteotoxic stress. This activation of HSF1 depended on dysregulated MAPK signaling. HSF1, in turn, supported MAPK signaling. In mice, Hsf1 deficiency impeded NF1-associated carcinogenesis by attenuating oncogenic RAS/MAPK signaling. In cell lines from human malignant peripheral nerve sheath tumors (MPNSTs) driven by NF1 loss, HSF1 was overexpressed and activated, which was required for tumor cell viability. In surgical resections of human MPNSTs, HSF1 was overexpressed, translocated to the nucleus, and phosphorylated. These findings reveal a surprising biological consequence of NF1 deficiency: activation of HSF1 and ensuing addiction to this master regulator of the heat shock response. The loss of NF1 function engages an evolutionarily conserved cellular survival mechanism that ultimately impairs survival of the whole organism by facilitating carcinogenesis. IntroductionEvolutionarily conserved from yeasts to humans, the heat shock transcription factor heat shock factor 1 (HSF1) is activated by a broad range of stressors that extend far beyond heat, including heavy metals, UV radiation, hypoxia, desiccation, and acidosis (1). During activation, HSF1 undergoes phosphorylation and other posttranslational modifications, trimerization, and nuclear translocation. This results in rapid, high-affinity binding of HSF1 to consensus heat shock elements (HSEs) within the promoters of target genes (2). Such binding drives the induction or suppression of hundreds of genes in mammalian cells (3).The adaptive response unleashed by HSF1 activation is critical for maintaining homeostasis of the cell's proteome, mediated in large part by increased expression of classical heat shock proteins such as HSP27, HSP70, and HSP90 (4). However, the effect of HSF1 activation goes far beyond these chaperones. It helps coordinate a range of fundamental cellular processes that are important to the fitness of malignant cells, including cell cycle control, ribosome biogenesis, protein translation, and glucose metabolism (5, 6). As a result, HSF1 both facilitates initial oncogenic transformation and maintains the malignant phenotype of established cancer cell lines driven by a wide range of mutations. In mice and in cell culture, genetic ablation of Hsf1 expression potently impairs tumorigenesis and cellular transformation driven by oncogene activation or tumor suppressor loss (5). The importance of HSF1 in enabling malignancy has been demonstrated by other recent work as well

To cope with proteotoxic stress, cells attenuate protein synthesis. However, the precise mechanisms underlying this fundamental adaptation remain poorly defined. Here we report that mTORC1 acts as an immediate cellular sensor of proteotoxic stress. Surprisingly, the multifaceted stress-responsive kinase JNK constitutively associates with mTORC1 under normal growth conditions. Upon activation by proteotoxic stress, JNK phosphorylates both RAPTOR at Ser863 and mTOR at Ser567, causing partial disintegration of mTORC1 and subsequent translation inhibition. Importantly, HSF1, the central player in the proteotoxic stress response (PSR), preserves mTORC1 integrity and function by inactivating JNK, independently of its canonical transcriptional action. Thereby, HSF1 translationally augments the PSR. Beyond promoting stress resistance, this intricate HSF1-JNK-mTORC1 interplay, strikingly, regulates cell, organ and body sizes. Thus, these results illuminate a unifying mechanism that controls stress adaptation and growth.

Numerous extrinsic and intrinsic insults trigger the HSF1-mediated proteotoxic stress response (PSR), an ancient transcriptional program that is essential to proteostasis and survival under such conditions. In contrast to its well-recognized mobilization by proteotoxic stress, little is known about how this powerful adaptive mechanism reacts to other stresses. Surprisingly, we discovered that metabolic stress suppresses the PSR. This suppression is largely mediated through the central metabolic sensor AMPK, which physically interacts with and phosphorylates HSF1 at Ser121. Through AMPK activation, metabolic stress represses HSF1, rendering cells vulnerable to proteotoxic stress. Conversely, proteotoxic stress inactivates AMPK and thereby interferes with the metabolic stress response. Importantly, metformin, a metabolic stressor and popular anti-diabetic drug, inactivates HSF1 and provokes proteotoxic stress within tumor cells, thereby impeding tumor growth. Thus, these findings uncover a novel interplay between the metabolic stress sensor AMPK and the proteotoxic stress sensor HSF1 that profoundly impacts stress resistance, proteostasis, and malignant growth.