The Reprogramming of Tumor Stroma by HSF1 Is a Potent Enabler of Malignancy

Stromal cells within the tumor microenvironment are essential for tumor progression and metastasis. Surprisingly little is known about the factors that drive the transcriptional reprogramming of stromal cells within tumors. We report that the transcriptional regulator Heat-Shock Factor 1 (HSF1) is frequently activated in cancer-associated fibroblasts (CAFs), where it is a potent enabler of malignancy. HSF1 drives a transcriptional program in CAFs that complements, yet is completely different from, the program it drives in adjacent cancer cells. This CAF program is uniquely structured to support the malignant potential of cancer cells in a non-cell-autonomous way. Two central stromal signaling molecules—TGFβ and stromal-derived factor 1 (SDF1) – play a critical role. In early stage breast and lung cancer, high stromal HSF1 activation is strongly associated with poor patient outcome. Thus, tumors co-opt the ancient survival functions of HSF1 to orchestrate malignancy in both cell-autonomous and non-cell-autonomous ways, with far-reaching therapeutic implications

Heat Shock Factor 1-dependent extracellular matrix remodeling mediates the transition from chronic intestinal inflammation to colon cancer

In the colon, long-term exposure to chronic inflammation drives colitis-associated colon cancer (CAC) in patients with inflammatory bowel disease. While the causal and clinical links are well established, molecular understanding of how chronic inflammation leads to the development of colon cancer is lacking. Here we deconstruct the evolving microenvironment of CAC by measuring proteomic changes and extracellular matrix (ECM) organization over time in a mouse model of CAC. We detect early changes in ECM structure and composition, and report a crucial role for the transcriptional regulator heat shock factor 1 (HSF1) in orchestrating these events. Loss of HSF1 abrogates ECM assembly by colon fibroblasts in cell-culture, prevents inflammation-induced ECM remodeling in mice and inhibits progression to CAC. Establishing relevance to human disease, we find high activation of stromal HSF1 in CAC patients, and detect the HSF1-dependent proteomic ECM signature in human colorectal cancer. Thus, HSF1-dependent ECM remodeling plays a crucial role in mediating inflammation-driven colon cancer.R35 GM118173 - NIGMS NIH HHS; U01 TR002625 - NCATS NIH HHS; P30 CA008748 - NCI NIH HHS; FC010144 - Cancer Research UK; FC010144 - Medical Research Council; FC010144 - Wellcome TrustPublished versio

First virtual international congress on cellular and organismal stress responses, november 5–6, 2020

Members of the Cell Stress Society International (CSSI), Patricija van Oosten-Hawle (University of Leeds, UK), Mehdi Mollapour (SUNY Upstate Medical University, USA), Andrew Truman (University of North Carolina at Charlotte, USA) organized a new virtual meeting format which took place on November 5–6, 2020. The goal of this congress was to provide an international platform for scientists to exchange data and ideas among the Cell Stress and Chaperones community during the Covid-19 pandemic. Here we will highlight the summary of the meeting and acknowledge those who were honored by the CSSI

Second Virtual International Symposium on Cellular and Organismal Stress Responses, September 8–9, 2022 [Meeting Review]

The Second International Symposium on Cellular and Organismal Stress Responses took place virtually on September 8–9, 2022. This meeting was supported by the Cell Stress Society International (CSSI) and organized by Patricija Van Oosten-Hawle and Andrew Truman (University of North Carolina at Charlotte, USA) and Mehdi Mollapour (SUNY Upstate Medical University, USA). The goal of this symposium was to continue the theme from the initial meeting in 2020 by providing a platform for established researchers, new investigators, postdoctoral fellows, and students to present and exchange ideas on various topics on cellular stress and chaperones. We will summarize the highlights of the meeting here and recognize those that received recognition from the CSSI

BRCA mutational status shapes the stromal microenvironment of pancreatic cancer linking clusterin expression in cancer associated fibroblasts with HSF1 signaling

Tumors initiate by mutations in cancer cells, and progress through interactions of the cancer cells with non-malignant cells of the tumor microenvironment. Major players in the tumor microenvironment are cancer-associated fibroblasts (CAFs), which support tumor malignancy, and comprise up to 90% of the tumor mass in pancreatic cancer. CAFs are transcriptionally rewired by cancer cells. Whether this rewiring is differentially affected by different mutations in cancer cells is largely unknown. Here we address this question by dissecting the stromal landscape of BRCA-mutated and BRCA Wild-type pancreatic ductal adenocarcinoma. We comprehensively analyze pancreatic cancer samples from 42 patients, revealing different CAF subtype compositions in germline BRCA-mutated vs. BRCA Wild-type tumors. In particular, we detect an increase in a subset of immune-regulatory clusterin-positive CAFs in BRCA-mutated tumors. Using cancer organoids and mouse models we show that this process is mediated through activation of heat-shock factor 1, the transcriptional regulator of clusterin. Our findings unravel a dimension of stromal heterogeneity influenced by germline mutations in cancer cells, with direct implications for clinical research

Deconstructing tumor heterogeneity: the stromal perspective.

Significant advances have been made towards understanding the role of immune cell-tumor interplay in either suppressing or promoting tumor growth, progression, and recurrence, however, the roles of additional stromal elements, cell types and/or cell states remain ill-defined. The overarching goal of this NCI-sponsored workshop was to highlight and integrate the critical functions of non-immune stromal components in regulating tumor heterogeneity and its impact on tumor initiation, progression, and resistance to therapy. The workshop explored the opposing roles of tumor supportive versus suppressive stroma and how cellular composition and function may be altered during disease progression. It also highlighted microenvironment-centered mechanisms dictating indolence or aggressiveness of early lesions and how spatial geography impacts stromal attributes and function. The prognostic and therapeutic implications as well as potential vulnerabilities within the heterogeneous tumor microenvironment were also discussed. These broad topics were included in this workshop as an effort to identify current challenges and knowledge gaps in the field

Challenging Proteostasis: Role of the Chaperone Network to Control Aggregation-Prone Proteins in Human Disease

Protein homeostasis (Proteostasis) is essential for correct and efficient protein function within the living cell. Among the critical components of the Proteostasis Network (PN) are molecular chaperones that serve widely in protein biogenesis under physiological conditions, and prevent protein misfolding and aggregation enhanced by conditions of cellular stress. For Alzheimer’s, Parkinson’s, Huntington’s diseases and ALS, multiple classes of molecular chaperones interact with the highly aggregation-prone proteins amyloid-β, tau, α-synuclein, huntingtin and SOD1 to influence the course of proteotoxicity associated with these neurodegenerative diseases. Accordingly, overexpression of molecular chaperones and induction of the heat shock response have been shown to be protective in a wide range of animal models of these diseases. In contrast, for cancer cells the upregulation of chaperones has the undesirable effect of promoting cellular survival and tumor growth by stabilizing mutant oncoproteins. In both situations, physiological levels of molecular chaperones eventually become functionally compromised by the persistence of misfolded substrates, leading to a decline in global protein homeostasis and the dysregulation of diverse cellular pathways. The phenomenon of chaperone competition may underlie the broad pathology observed in aging and neurodegenerative diseases, and restoration of physiological protein homeostasis may be a suitable therapeutic avenue for neurodegeneration as well as for cancer

AmB-resistant strains are hypersensitive to the stresses of the host environment.

<p>(A) AmB-resistant strains are sensitive to very high febrile temperatures and extremely sensitive to oxidative stress, especially at elevated temperature. Wild-type and AmB-resistant strains were spotted by serial dilution on RPMI media with or without tert-butyl peroxide at the concentrations indicated. (B–E) AmB-resistant strains are hypersensitive to stresses encountered in the host environment. Wild-type and AmB-resistant strains were grown in RPMI media containing 2 mM of hypochlorous acid (B) or 4 mM of the nitric oxide donor DPTA-NONOATE (C), two neutrophil-secreted products that are the critical final effectors of anti-<i>Candida</i> immunity. Sensitivity to iron deprivation was tested by growth in RPMI+500 µM of the iron chelator bathophenanthrolinedisulfonic acid (D). No increase in sensitivity to the antimicrobial peptide Calprotectin (10 µg/mL) (E) was observed. Growth values were obtained by normalization to wild-type growth in the same condition. All mutant strains were significantly more sensitive than wild-type to the tested concentrations of hypochlorous acid, DPTA-NONOate, and BPS at 37°C (**<i>p</i><0.01, two-tailed Student's <i>t</i> test), but not to calprotectin. Values indicate the mean of two independent experiments of three replicates each; error bars indicate SEM. (F) AmB-resistant strains proliferate more slowly than wild type in serum at elevated temperature. Wild-type and AmB-resistant strains were inoculated in 100% fetal bovine serum at 39.5°C, and viable colony-forming units were determined by plating dilutions on YPD after 24 and 48 h. Error bars indicate SEM. (G) AmB-resistant strains are hypersensitive to killing by neutrophils. Human neutrophils were isolated from whole blood (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001692#s4" target="_blank">Materials and Methods</a>) and activated by treatment with recombinant TNF-α. <i>Candida</i> were added to wells containing neutrophils at a 1∶1 effector∶target ratio and incubated at 37°C for 6 h, at which point neutrophils were lysed and <i>Candida</i> growth was quantitated with Alamar blue. Percent growth for each strain was calculated as the fraction of growth in the presence of neutrophils to growth in the absence of neutrophils; mutants were normalized to growth of the WT on each plate. The experiment was performed with a total of three biological replicates from two separate days; error bars indicate mean and SEM.</p

Constitutive stress response activation in AmB-resistant strains.

<p>(A) AmB resistant mutants constitutively express diverse stress response genes at high levels in the absence of any external stressors. qRT-PCR profiling of stress response genes in wild-type, fluconazole-resistant (<i>erg3</i>), and AmB-resistant mutants of <i>C. albicans</i> grown in rich (YPD) media with no added stressors (left panel), compared with wild-type strains treated with a variety of acute stressors known to activate these stress response pathways (right panel). Strains were grown to mid-log phase in YPD, and RNA was isolated by established methods. For stress treatment of wild-type strains (right panel), the indicated stressors are: AmB, 1 µg/mL AmB; NaCl, 0.3 M NaCl; Fe Chelator, 500 µM bapthophenanthroline disulfonate; DPTA-NO, 2 mM DPTA-NONOate; Ca²⁺, 150 mM CaCl₂; Peroxide, 10 mM tert-butyl peroxide. Expression levels of diverse stress response genes were quantified and normalized to four internal control genes: TDH3, TEF3, ACT1, and RPP2B; the mean value obtained from these four normalizations was used. To generate quantitative comparisons for color visualization, the relative expression level of each gene in each sample was divided by its maximal expression level observed (in any of the deletion strains or stress conditions; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001692#s4" target="_blank">Materials and Methods</a> for further description). (B) Calcineurin and PKC pathways are differentially required for survival of AmB-resistant strains. Growth of AmB-resistant laboratory mutants and clinical isolates, as well as wild-type controls, in the presence of small molecule stress response inhibitors at the following concentrations: Geldanamycin and Radicicol, 2 µM; FK-506, 5 µg/mL; Cyclosporin A, 5 µg/mL; Enzastaurin and Cercosporamide, 5 µg/mL. Growth was assayed after 24 h; values indicate means of duplicate measurements, normalized to wild-type in DMSO. (C) Hog1, but not calcineurin, is required for wild-type levels of AmB tolerance in the absence of ergosterol biosynthesis mutations. Wild-type, <i>cnb1</i>, and <i>hog1</i> strains, as well as wild-type with 1 or 5 µM geldanamycin, were tested for AmB MIC by microplate dilution. Growth was assayed after 48 h to highlight differences in drug tolerance between strains; values indicate means of duplicate measurements, normalized to the wild type in the absence of AmB. (D) Hog1, Cnb1, and high levels of Hsp90 are required for the <i>de novo</i> emergence of AmB-resistant colonies. <i>ERG2/erg2</i> heterozygotes from wild-type, <i>cnb1</i>, or <i>hog1</i> backgrounds were plated at a density of 8×10⁶ cells per plate on media containing 0.4 µg/mL AmB. The wild-type was also plated on media containing AmB and 2.5 µM geldanamycin. Plates were photographed after 2 d.</p

AmB-resistant strains critically depend on high levels of Hsp90 function for survival.

<p>(A) Mild inhibition of Hsp90 not only reverses AmB resistance but selectively kills AmB-resistant isolates. Spot assays (5-fold serial dilutions) of wild-type (SC5314), fluconazole-resistant (Isolate #2, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001692#pbio.1001692-White2" target="_blank">[61]</a>), and AmB-resistant (ATCC 200955) <i>C. albicans</i> on RPMI media containing antifungals and/or Hsp90 inhibitors at the indicated concentrations. (B) Increased dependence on Hsp90 for AmB resistance is conserved in a <i>C. tropicalis</i> clinical isolate. Spot assays of <i>C. tropicalis</i> reference strain MYA-3404 (AmB-sensitive) and ATCC 200956 (AmB-resistant) on media containing AmB, geldanamycin, or both compounds. (C) Isogenic, laboratory-generated AmB-resistant mutants also show an increased dependence on Hsp90. Spot assay of laboratory-generated mutants on AmB and geldanamycin. Fluconazole-resistant <i>erg3</i> mutant and AmB-resistant clinical isolate are provided for reference. Full MIC data in liquid culture for all strains are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001692#pbio.1001692.s004" target="_blank">Figure S4</a>. (D) <i>In vitro</i> selection for AmB resistance leads to hypersensitivity to Hsp90 inhibition. 24-hour MIC80 (drug concentration reducing growth by 80 percent) of geldanamycin, radicicol, and AmB for each isolate from <i>in vitro</i> evolution series, tested in YPD at 30°C. Note the discontinuity in the left <i>y</i>-axis due to the dramatic decrease in Hsp90 inhibitor MIC. (E) Hsp90 inhibition is cidal to AmB-resistant strains. Viability assays of wild-type and AmB-resistant <i>C. albicans</i> strains in the presence of the Hsp90 inhibitors geldanamycin or radicicol. Strains were incubated in liquid culture for 24 h with the drugs and then plated for surviving colony-forming units. Drugs were used at 5 µM in all strains. Error bars indicate SEM; each measurement was performed in duplicate.</p