Martina Koeva

Quantitative Analysis of Hsp90-Client Interactions Reveals Principles of Substrate Recognition

HSP90 is a molecular chaperone that associates with numerous substrate proteins called clients. It plays many important roles in human biology and medicine, but determinants of client recognition by HSP90 have remained frustratingly elusive. We systematically and quantitatively surveyed most human kinases, transcription factors, and E3 ligases for interaction with HSP90 and its cochaperone CDC37. Unexpectedly, many more kinases than transcription factors bound HSP90. CDC37 interacted with kinases, but not with transcription factors or E3 ligases. HSP90::kinase interactions varied continuously over a 100-fold range and provided a platform to study client protein recognition. In wild-type clients, HSP90 did not bind particular sequence motifs, but rather associated with intrinsically unstable kinases. Stabilization of the kinase in either its active or inactive conformation with diverse small molecules decreased HSP90 association. Our results establish HSP90 client recognition as a combinatorial process: CDC37 provides recognition of the kinase family, whereas thermodynamic parameters determine client binding within the family.

Tight Coordination of Protein Translation and HSF1 Activation Supports the Anabolic Malignant State

Introduction Ribosome biogenesis is commonly up-regulated to satisfy the increased anabolic demands associated with malignant transformation and tumor growth. Many different oncogenic signaling pathways converge on the ribosome to increase translational flux. Despite the detailed understanding of ribosome regulation in cancer, it is not clear whether the net translational activity of the ribosome can itself regulate transcriptional programs that support and promote the malignant state. Methods To investigate the transcriptional effects of modulating translational activity in malignant cells, we used integrated chemical and genetic approaches, including a gene signature–based genetic and chemical screen of more than 600,000 gene expression profiles (LINCS database) and an independent, reporter-based chemical screen of more than 300,000 compounds. A lead compound was tested in several cell-lines unified by their increased dependence on HSF1 activation for growth and survival, and in an in vivo cancer model. Results Inhibiting translation led to large changes in the transcriptome. The single most enriched category consisted of genes regulated by the heat-shock transcription factor, HSF1. The most down-regulated mRNA was HSPA8, which encodes the constitutive HSP70 chaperone that helps to fold nascent polypeptides. The expression of many other genes that HSF1 coordinates to support cancer were also strongly affected. HSF1 protein levels were unchanged, but HSF1 DNA occupancy was nearly eliminated. Inhibition of the HSF1-regulated gene expression program is thus a dominant transcriptional effect elicited by inhibiting protein translation. Using a gene signature of HSF1 inactivation to query the LINCS database revealed a strong connection between HSF1 inactivation and perturbations that inhibit protein translation, including a broad spectrum of chemical and genetic interventions that target the ribosome, eukaryotic initiation factors (eIFs), aminoacyl tRNA synthetases, and upstream signaling/regulatory pathways that control translation. Our high-throughput small-molecule screen identified rocaglamide A, an inhibitor of translation initiation, was the strongest inhibitor of HSF1 activation. An analog of this compound, RHT, increased thioredoxin-interacting protein (TXNIP) mRNA and protein levels and decreased glucose uptake and lactate production. Cell-based cancer models characterized by high dependence on HSF1 activation for growth and survival were highly sensitive to RHT, as were cells derived from diverse hematopoietic malignancies. RHT had a strong antitumor effect—with marked inhibition of HSF1 activity and glucose uptake—against xenografted acute myeloid leukemia cells. Discussion The ribosome functions as a central information hub in malignant cells: Translational flux conveys information about the cell’s metabolic status to regulate the transcriptional programs that support it. Multiple unbiased chemical and genetic approaches establish HSF1 as a prime transducer of this information, centrally poised to regulate the transcription of genes that support protein folding, biomass expansion, anabolic metabolism, cellular proliferation, and survival. Targeting translation initiation may offer a strategy for reversing HSF1 activation, disabling metabolic and cytoprotective pathways in malignant cells. HSF1 at the crossroads of protein translation and metabolism. (Left) Cancers activate an HSF1-regulated transcriptional program to adapt to the anabolic demands of relentless biomass expansion. Glucose uptake increases, and expression of TXNIP, an inhibitor of glucose uptake, drops. (Right) Down-regulating translation with rocaglate scaffold initiation inhibitors reverses cancer-associated HSF1 activation. Glucose uptake drops as TXNIP levels rise. Sensing Reduced Translation The interplay between metabolic pathways and the cellular survival programs that enable tumors to grow are poorly understood. Heat shock factor 1 (HSF1) coordinates an unexpectedly diverse transcriptional network involved in oncogenesis. Santagata et al. (p. 1238303; see the Perspective by Gandin and Topisirovic) found that reduced translation may be used to sense a cells metabolic status and regulate transcription, in particular by inactivating HSF1 with consequent affects on its targets. Small-molecule drugs that affected this link were able to inhibit the growth of transformed cells in culture and of an animal tumor model. Chemical and genetic screening links ribosome activity levels and a transcriptional regulator in malignant cells. [Also see Perspective by Gandin and Topisirovic] The ribosome is centrally situated to sense metabolic states, but whether its activity, in turn, coherently rewires transcriptional responses is unknown. Here, through integrated chemical-genetic analyses, we found that a dominant transcriptional effect of blocking protein translation in cancer cells was inactivation of heat shock factor 1 (HSF1), a multifaceted transcriptional regulator of the heat-shock response and many other cellular processes essential for anabolic metabolism, cellular proliferation, and tumorigenesis. These analyses linked translational flux to the regulation of HSF1 transcriptional activity and to the modulation of energy metabolism. Targeting this link with translation initiation inhibitors such as rocaglates deprived cancer cells of their energy and chaperone armamentarium and selectively impaired the proliferation of both malignant and premalignant cells with early-stage oncogenic lesions.

Poly-glutamine expanded huntingtin dramatically alters the genome wide binding of HSF1.

In Huntingtons disease (HD), polyglutamine expansions in the huntingtin (Htt) protein cause subtle changes in cellular functions that, over-time, lead to neurodegeneration and death. Studies have indicated that activation of the heat shock response can reduce many of the effects of mutant Htt in disease models, suggesting that the heat shock response is impaired in the disease. To understand the basis for this impairment, we have used genome-wide chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) to examine the effects of mutant Htt on the master regulator of the heat shock response, HSF1. We find that, under normal conditions, HSF1 function is highly similar in cells carrying either wild-type or mutant Htt. However, polyQ-expanded Htt severely blunts the HSF1-mediated stress response. Surprisingly, we find that the HSF1 targets most affected upon stress are not directly associated with proteostasis, but with cytoskeletal binding, focal adhesion and GTPase activity. Our data raise the intriguing hypothesis that the accumulated damage from life-long impairment in these stress responses may contribute significantly to the etiology of Huntingtons disease.

Network Modeling of Heterogeneous Datasets

The cellular environment is a complex and dynamic system of functional molecules. These molecules interact, either transiently or stably, in pathways, which remain poorly mapped despite extensive study. High-throughput experimental methods have allowed us to fill some of the gaps in pathway knowledge, but have created new algorithmic challenges associated with the integration of large, disparate sources of data. Here, we present different algorithmic approaches to the integration of heterogeneous datasets. The article examines algorithms that analyze networks with a single data source, methods that analyze networks with more complex data types, and finally algorithms that capture hierarchical flow of biological information. We also examine a critical issue that has been largely ignored in most network approaches: To what extent can gene expression data be used as a proxy for protein levels? While most studies conflate these two, it is now abundantly clear from high-throughput analyses that the correlation between these data is very poor. We close by speculating on where a more realistic modeling of different types of data will lead the field.

Abstract A23: Cell autonomous and nonautonomous activities of heat shock factor 1 support tumor initiation, progression, and metastasis

The heat-shock response is a powerful transcriptional program which acts genome-wide, not only to restore the normal protein folding through the induction of heat shock proteins (HSP), but to re-shape global cellular pathways controlling survival, growth and metabolism. In mammals, this response is regulated primarily by the Heat Shock Factor 1 (HSF1) transcription factor. We have previously shown that HSF1 plays a fundamental role in tumorigenesis, by promoting the survival and malignance of tumor cells, both in tissue culture and in mouse models of cancer [1]. Recently we demonstrated that HSF1 exerts its role by activating a unique transcriptional program in the cancer cells, that is distinct from the one activated during heat shock [2]. In breast cancer and several other types of carcinoma, we found that high HSF1 protein levels and activation of the HSF1-dependent transcriptional program are associated with poor clinical outcome [3]. Here we show that HSF1 is activated not only in the tumor cells, but also in the stromal cells infiltrating the tumor. Examining human patient samples, we find immunohistochemical evidence for activation of HSF1 in the stroma. Using mouse xenograft models and in vitro co-culture we show that HSF1 in the stroma supports tumor cell growth. Finally, expression profiling and analysis of the DNA binding pattern of HSF1 in tumors and in cell culture indicates that stromal HSF1 supports tumorigenesis by activating a unique, stroma-specific transcriptional program. Taken together, our data suggests that HSF1 acts in the cancer cells and in the stroma to activate distinct, yet complimentary transcriptional programs that will facilitate tumor initiation, progression and metastasis. References 1. Dai, C., et al., Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis. Cell, 2007. 130(6): p. 1005-18. 2. Mendillo, M.L., et al., HSF1 drives a transcriptional program distinct from heat shock to support highly malignant human cancers. Cell, 2012. 150(3): p. 549-62. 3. Santagata, S., et al., High levels of nuclear heat-shock factor 1 (HSF1) are associated with poor prognosis in breast cancer. Proc Natl Acad Sci U S A, 2011. 108(45): p. 18378-83. Citation Format: Ruth Scherz-Shouval, Sandro Santagata, Martina Koeva, Luke Whitesell, Susan Lindquist. Cell autonomous and nonautonomous activities of heat shock factor 1 support tumor initiation, progression, and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A23.

P3-01-09: Oncogenic Activation of HSF1 Enables the Malignant Progression of Breast Carcinoma.

HSF1 is best known for its role as the master transcriptional regulator of the evolutionarily conserved heat-shock response. In mice, Hsf1 knock-outs dramatically reduce susceptibility to malignant transformation and tumor formation, and markedly increased survival in cancers driven by both oncogenic and tumor suppressor mutations. Likewise, RNAi-mediated knockdown markedly reduces the growth and survival of human cell lines established from cancers driven by a diversity of genetic lesions. The transcriptional network that HSF1 coordinates during heat-shock is known, but is unknown in malignancy. We compare HSF1 function in isogenic breast cancer cells of high and low malignant potential. HSF1 orchestrates a far-reaching transcriptional program that is dependent on transformation and the degree of malignancy. The oncogenic HSF1 program differs markedly from the classical heat-shock response. It is enriched for genes involved in a myriad of cellular processes known to be important in malignancy, including transcription, translation, glucose metabolism and cellular adhesion. It includes only a particular subset of heat-shock protein genes, and many of these are regulated in a manner that differs from their regulation by heat-shock. We also find that HSF1 is overexpressed and activated in a large subset of all conventionally defined classes of breast cancer. Moreover, tumors with high expression of the oncogenic HSF1 transcriptional network are strongly associated with poor outcome as monitored by metastasis and death. Our findings suggest that the oncogenic HSF1 activation program is rooted in fundamental aspects of tumor biology and will prove a powerful new tool in the clinical management of patients with breast cancer and, likely, other malignancies as well. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-09.