Marc L. Mendillo

High levels of nuclear heat-shock factor 1 (HSF1) are associated with poor prognosis in breast cancer

Heat-shock factor 1 (HSF1) is the master transcriptional regulator of the cellular response to heat and a wide variety of other stressors. We previously reported that HSF1 promotes the survival and proliferation of malignant cells. At this time, however, the clinical and prognostic significance of HSF1 in cancer is unknown. To address this issue breast cancer samples from 1,841 participants in the Nurses’ Health Study were scored for levels of nuclear HSF1. Associations of HSF1 status with clinical parameters and survival outcomes were investigated by Kaplan–Meier analysis and Cox proportional hazard models. The associations were further delineated by Kaplan–Meier analysis using publicly available mRNA expression data. Our results show that nuclear HSF1 levels were elevated in ∼80% of in situ and invasive breast carcinomas. In invasive carcinomas, HSF1 expression was associated with high histologic grade, larger tumor size, and nodal involvement at diagnosis (P < 0.0001). By using multivariate analysis to account for the effects of covariates, high HSF1 levels were found to be independently associated with increased mortality (hazards ratio: 1.62; 95% confidence interval: 1.21–2.17; P < 0.0013). This association was seen in the estrogen receptor (ER)-positive population (hazards ratio: 2.10; 95% confidence interval: 1.45–3.03; P < 0.0001). In public expression profiling data, high HSF1 mRNA levels were also associated with an increase in ER-positive breast cancer-specific mortality. We conclude that increased HSF1 is associated with reduced breast cancer survival. The findings indicate that HSF1 should be evaluated prospectively as an independent prognostic indicator in ER-positive breast cancer. HSF1 may ultimately be a useful therapeutic target in cancer.

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.

A Genome-wide siRNA Screen Identifies Proteasome Addiction as a Vulnerability of Basal-like Triple-Negative Breast Cancer Cells

Basal-like triple-negative breast cancers (TNBCs) have poor prognosis. To identify basal-like TNBC dependencies, a genome-wide siRNA lethality screen compared two human breast epithelial cell lines transformed with the same genes: basal-like BPLER and myoepithelial HMLER. Expression of the screens 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Proteasome genes were overrepresented hits. Basal-like TNBC lines were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal, and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiating cell function and macrometastasis. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence.

A conserved MutS homolog connector domain interface interacts with MutL homologs

Escherichia coli MutS forms a mispair-dependent ternary complex with MutL that is essential for initiating mismatch repair (MMR) but is structurally uncharacterized, in part owing to its dynamic nature. Here, we used hydrogen/deuterium exchange mass spectrometry and other methods to identify a region in the connector domain (domain II) of MutS that binds MutL and is required for mispair-dependent ternary complex formation and MMR. A structurally conserved region in Msh2, the eukaryotic homolog, was required for formation of a mispair-dependent Msh2–Msh6–Mlh1–Pms1 ternary complex. These data indicate that the connector domain of MutS and Msh2 contains the interface for binding MutL and Mlh1–Pms1, respectively, and support a mechanism whereby mispair and ATP binding induces a conformational change that allows the MutS and Msh2 interfaces to interact with their partners.

Precise genetic mapping and haplotype analysis of the familial dysautonomia gene on human chromosome 9q31.

Familial dysautonomia (FD) is an autosomal recessive disorder characterized by developmental arrest in the sensory and autonomic nervous systems and by Ashkenazi Jewish ancestry. We previously had mapped the defective gene (DYS) to an 11-cM segment of chromosome 9q31-33, flanked by D9S53 and D9S105. By using 11 new polymorphic loci, we now have narrowed the location of DYS to <0.5 cM between the markers 43B1GAGT and 157A3. Two markers in this interval, 164D1 and D9S1677, show no recombination with the disease. Haplotype analysis confirmed this candidate region and revealed a major haplotype shared by 435 of 441 FD chromosomes, indicating a striking founder effect. Three other haplotypes, found on the remaining 6 FD chromosomes, might represent independent mutations. The frequency of the major FD haplotype in the Ashkenazim (5 in 324 control chromosomes) was consistent with the estimated DYS carrier frequency of 1 in 32, and none of the four haplotypes associated with FD was observed on 492 non-FD chromosomes from obligatory carriers. It is now possible to provide accurate genetic testing both for families with FD and for carriers, on the basis of close flanking markers and the capacity to identify >98% of FD chromosomes by their haplotype.

Escherichia coli MutS Tetramerization Domain Structure Reveals That Stable Dimers but Not Tetramers Are Essential for DNA Mismatch Repair in Vivo

The Escherichia coli mispair-binding protein MutS forms dimers and tetramers in vitro, although the functional form in vivo is under debate. Here we demonstrate that the MutS tetramer is extended in solution using small angle x-ray scattering and the crystal structure of the C-terminal 34 amino acids of MutS containing the tetramer-forming domain fused to maltose-binding protein (MBP). Wild-type C-terminal MBP fusions formed tetramers and could bind MutS and MutS-MutL-DNA complexes. In contrast, D835R and R840E mutations predicted to disrupt tetrameric interactions only allowed dimerization of MBP. A chromosomal MutS truncation mutation eliminating the dimerization/tetramerization domain eliminated mismatch repair, whereas the tetramer-disrupting MutS D835R and R840E mutations only modestly affected MutS function. These results demonstrate that dimerization but not tetramerization of the MutS C terminus is essential for mismatch repair.

HSP90 empowers evolution of resistance to hormonal therapy in human breast cancer models

Significance Although hormonal therapies for estrogen receptor-positive (ER+) breast cancer make up the earliest, and arguably most effective, “molecularly targeted” anticancer drugs, continued progress in controlling metastatic disease has been slow. Heterogeneity and the complexity of signaling in advanced cancers have frustrated efforts to prevent the rapid evolution of resistance to hormonal therapies, as well as kinase inhibitors and other agents. On the basis of earlier work defining the role of heat shock protein 90 (HSP90) in other evolutionary processes, we tested whether low-level HSP90 inhibition would limit the evolution of hormone resistance in breast cancer models. Results in culture and in mice provide support for a readily implemented strategy by which the heterogeneity and evolvability of metastatic ER+ breast tumors, and perhaps other advanced cancers, might be controlled. The efficacy of hormonal therapies for advanced estrogen receptor-positive breast cancers is limited by the nearly inevitable development of acquired resistance. Efforts to block the emergence of resistance have met with limited success, largely because the mechanisms underlying it are so varied and complex. Here, we investigate a new strategy aimed at the very processes by which cancers evolve resistance. From yeast to vertebrates, heat shock protein 90 (HSP90) plays a unique role among molecular chaperones by promoting the evolution of heritable new traits. It does so by regulating the folding of a diverse portfolio of metastable client proteins, many of which mediate adaptive responses that allow organisms to adapt and thrive in the face of diverse challenges, including those posed by drugs. Guided by our previous work in pathogenic fungi, in which very modest HSP90 inhibition impairs resistance to mechanistically diverse antifungals, we examined the effect of similarly modest HSP90 inhibition on the emergence of resistance to antiestrogens in breast cancer models. Even though this degree of inhibition fell below the threshold for proteotoxic activation of the heat-shock response and had no overt anticancer activity on its own, it dramatically impaired the emergence of resistance to hormone antagonists both in cell culture and in mice. Our findings strongly support the clinical testing of combined hormone antagonist-low-level HSP90 inhibitor regimens in the treatment of metastatic estrogen receptor-positive breast cancer. At a broader level, they also provide promising proof of principle for a generalizable strategy to combat the pervasive problem of rapidly emerging resistance to molecularly targeted therapeutics.

Coupling distant sites in DNA during DNA mismatch repair

How can the activities of different proteins that act at two distant sites on DNA be coupled? In cis mechanisms, an intact DNA helix is required so that the protein molecules can slide (diffuse), translocate, polymerize, or otherwise communicate along the DNA helix with the second site. Examples include the nucleotide-dependent translocation of type I and III restriction endonucleases (1) and the polymerization of SeqA that blocks bacterial replication origins (2). In trans mechanisms, contacts occur between the two sites, and the intervening DNA is looped out; this also could occur between sites on different DNAs. Trans mechanisms are common in the regulation of gene expression (3). The DNA mismatch repair (MMR) system must recognize mispairs resulting from replication errors, distinguish between newly synthesized and parental DNA strands, and coordinate an excision repair reaction so that the misincorporated base is removed from the newly synthesized strand (4). In Escherichia coli, MutL mediates communication between the mispairs, which are recognized by MutS, and a distant hemimethylated GATC site, which is cleaved by MutH on the newly synthesized strand that is not yet modified by the Dam methylase (Fig. 1). The single-strand break created by MutH can direct excision either 3′ or 5′ to the mispaired base and is the key postinitiation intermediate in MMR that allows excision to proceed by means of the UvrD helicase and one of four single-stranded DNA exonucleases. Efficient MutH cleavage depends on MutS, MutL, and the mispair; however, the mechanism by which this coupling occurs and drives appropriate directionality of excision is poorly understood even though E. coli MMR was first reconstituted with purified proteins almost 20 years ago (4). In a recent issue of PNAS, Pluciennik and Modrich (5) provided welcome insight into the problem; their results support a cis mechanism for MMR.

Luminidependens (LD) is an Arabidopsis protein with prion behavior

Significance Prion proteins provide the best-understood mode for protein-based molecular memory. Since their discovery in mammals, prions have been identified in diverse organisms including fungi, Aplysia, and Drosophila, but not in the plant kingdom. Applying methods we used to uncover yeast prions, we identified nearly 500 Arabidopsis proteins that harbor potential prion-like domains (PrDs). At least one of these domains, Luminidependens PrD, had some of the classical characteristics of prion proteins when tested experimentally in yeast, making it, to our knowledge, the first protein from the plant kingdom with bona fide prion attributes. Importantly, Luminidependens is involved in the process of flowering, a crucial development course that integrates several internal and external cues, including memories of winter, for its regulation. Prion proteins provide a unique mode of biochemical memory through self-perpetuating changes in protein conformation and function. They have been studied in fungi and mammals, but not yet identified in plants. Using a computational model, we identified candidate prion domains (PrDs) in nearly 500 plant proteins. Plant flowering is of particular interest with respect to biological memory, because its regulation involves remembering and integrating previously experienced environmental conditions. We investigated the prion-forming capacity of three prion candidates involved in flowering using a yeast model, where prion attributes are well defined and readily tested. In yeast, prions heritably change protein functions by templating monomers into higher-order assemblies. For most yeast prions, the capacity to convert into a prion resides in a distinct prion domain. Thus, new prion-forming domains can be identified by functional complementation of a known prion domain. The prion-like domains (PrDs) of all three of the tested proteins formed higher-order oligomers. Uniquely, the Luminidependens PrD (LDPrD) fully replaced the prion-domain functions of a well-characterized yeast prion, Sup35. Our results suggest that prion-like conformational switches are evolutionarily conserved and might function in a wide variety of normal biological processes.

Probing DNA- and ATP-mediated Conformational Changes in the MutS Family of Mispair Recognition Proteins Using Deuterium Exchange Mass Spectrometry

We have performed deuterium exchange mass spectrometry (DXMS) to probe the conformational changes that the bacterial MutS homodimer and the homologous eukaryotic heterodimer Msh2-Msh6 undergo when binding to ATP or DNA. The DXMS data support the view that high affinity binding to mispair-containing DNA and low affinity binding to fully base-paired DNA both involve forming rings by MutS protein family dimers around the DNA; however, mispair binding protects additional regions from deuterium exchange. DXMS also reveals two distinct conformations upon binding one or two ATP molecules and that binding of two ATP molecules propagates conformational changes to other regions of the protein complexes. The regions showing major changes in deuterium exchange upon ATP binding tend to occur in regions distinct from those involved in DNA binding, suggesting that although communication occurs between DNA and nucleotide binding, sliding clamps formed by binding both ATP and mispairs could result from the simultaneous action of two independent conformational changes.