Oncogenic KRASG12D Reprograms Lipid Metabolism by Upregulating SLC25A1 to Drive Pancreatic Tumorigenesis.

Pancreatic cancer is a highly lethal disease with obesity as one of the risk factors. Oncogenic KRAS mutations are prevalent in pancreatic cancer and can rewire lipid metabolism by altering fatty acid (FA) uptake, FA oxidation (FAO), and lipogenesis. Identification of the underlying mechanisms could lead to improved therapeutic strategies for treating KRAS-mutant pancreatic cancer. Here, we observed that KRASG12D upregulated the expression of SLC25A1, a citrate transporter that is a key metabolic switch to mediate FAO, fatty acid synthesis, glycolysis, and gluconeogenesis. In genetically engineered mouse models and human pancreatic cancer cells, KRASG12D induced SLC25A1 upregulation via GLI1, which directly stimulated SLC25A1 transcription by binding its promoter. The enhanced expression of SLC25A1 increased levels of cytosolic citrate, FAs, and key enzymes in lipid metabolism. In addition, a high-fat diet (HFD) further stimulated the KRASG12D-GLI1-SLC25A1 axis and the associated increase in citrate and FAs. Pharmacologic inhibition of SLC25A1 and upstream GLI1 significantly suppressed pancreatic tumorigenesis in KrasG12D/+ mice on a HFD. These results reveal a KRASG12D-GLI1-SLC25A1 regulatory axis, with SLC25A1 as an important node that regulates lipid metabolism during pancreatic tumorigenesis, thus indicating an intervention strategy for oncogenic KRAS-driven pancreatic cancer. SIGNIFICANCE: Upregulation of SLC25A1 induced by KRASG12D-GLI1 signaling rewires lipid metabolism and is exacerbated by HFD to drive the development of pancreatic cancer, representing a targetable metabolic axis to suppress pancreatic tumorigenesis.

Structure and function of the TPR-domain immunophilins FKBP51 and FKBP52 in normal physiology and disease.

Coordinated cochaperone interactions with Hsp90 and associated client proteins are crucial for a multitude of signaling pathways in normal physiology, as well as in disease settings. Research on the molecular mechanisms regulated by the Hsp90 multiprotein complexes has demonstrated increasingly diverse roles for cochaperones throughout Hsp90-regulated signaling pathways. Thus, the Hsp90-associated cochaperones have emerged as attractive therapeutic targets in a wide variety of disease settings. The tetratricopeptide repeat (TPR)-domain immunophilins FKBP51 and FKBP52 are of special interest among the Hsp90-associated cochaperones given their Hsp90 client protein specificity, ubiquitous expression across tissues, and their increasingly important roles in neuronal signaling, intracellular calcium release, peptide bond isomerization, viral replication, steroid hormone receptor function, and cell proliferation to name a few. This review summarizes the current knowledge of the structure and molecular functions of TPR-domain immunophilins FKBP51 and FKBP52, recent findings implicating these immunophilins in disease, and the therapeutic potential of targeting FKBP51 and FKBP52 for the treatment of disease.

Second Virtual International Symposium on Cellular and Organismal Stress Responses, September 8-9, 2022.

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.

Functions of the Hsp90-Binding FKBP Immunophilins.

The Hsp90 chaperone is known to interact with a diverse array of client proteins. However, in every case examined, Hsp90 is also accompanied by a single or several co-chaperone proteins. One class of co-chaperone contains a tetratricopeptide repeat (TPR) domain that targets the co-chaperone to the C-terminal region of Hsp90. Within this class are Hsp90-binding peptidylprolyl isomerases, most of which belong to the FK506-binding protein (FKBP) family. Despite the common association of FKBP co-chaperones with Hsp90, it is abundantly clear that the client protein influences, and is often influenced by, the particular FKBP bound to Hsp90. Examples include Xap2 in aryl hydrocarbon receptor complexes and FKBP52 in steroid receptor complexes. In this chapter, we discuss the known functional roles played by FKBP co-chaperones and, where possible, relate distinctive functions to structural differences between FKBP members.

Enhancing grant-writing expertise in BUILD institutions: Building infrastructure leading to diversity.

BACKGROUND: The lack of race/ethnic and gender diversity in grants funded by the National Institutes of Health (NIH) is a persistent challenge related to career advancement and the quality and relevance of health research. We describe pilot programs at nine institutions supported by the NIH-sponsored Building Infrastructure Leading to Diversity (BUILD) program aimed at increasing diversity in biomedical research. METHODS: We collected data from the 2016-2017 Higher Education Research Institute survey of faculty and NIH progress reports for the first four years of the program (2015-2018). We then conducted descriptive analyses of data from the nine BUILD institutions that had collected data and evaluated which activities were associated with research productivity. We used Poisson regression and rate ratios of the numbers of BUILD pilots funded, students included, abstracts, presentations, publications, and submitted and funded grant proposals. RESULTS: Teaching workshops were associated with more abstracts (RR 4.04, 95% CI 2.21-8.09). Workshops on grant writing were associated with more publications (RR 2.64, 95% CI 1.64-4.34) and marginally with marginally more presentations. Incentives to develop courses were associated with more abstracts published (RR 4.33, 95% CI 2.56-7.75). Workshops on research skills and other incentives were not associated with any positive effects. CONCLUSIONS: Pilot interventions show promise in supporting diversity in NIH-level research. Longitudinal modeling that considers time lags in career development in moving from project development to grants submissions can provide more direction for future diversity pilot interventions.

Building a Diverse Workforce and Thinkforce to Reduce Health Disparities.

The Research Centers in Minority Institutions (RCMI) Program was congressionally mandated in 1985 to build research capacity at institutions that currently and historically recruit, train, and award doctorate degrees in the health professions and health-related sciences, primarily to individuals from underrepresented and minority populations. RCMI grantees share similar infrastructure needs and institutional goals. Of particular importance is the professional development of multidisciplinary teams of academic and community scholars (the "workforce") and the harnessing of the heterogeneity of thought (the "thinkforce") to reduce health disparities. The purpose of this report is to summarize the presentations and discussion at the RCMI Investigator Development Core (IDC) Workshop, held in conjunction with the RCMI Program National Conference in Bethesda, Maryland, in December 2019. The RCMI IDC Directors provided information about their professional development activities and Pilot Projects Programs and discussed barriers identified by new and early-stage investigators that limit effective career development, as well as potential solutions to overcome such obstacles. This report also proposes potential alignments of professional development activities, targeted goals and common metrics to track productivity and success.

Bioanalytical Assay Development and Validation for the Pharmacokinetic Study of GMC1, a Novel FKBP52 Co-chaperone Inhibitor for Castration Resistant Prostate Cancer.

BACKGROUND: GMC1 (2-(1H-benzimidazol-2-ylsulfanyl)-N-[(Z)-(4-methoxyphenyl) methylideneamino] acetamide) effectively inhibits androgen receptor function by binding directly to FKBP52. This is a novel mechanism for the treatment of castration resistant prostate cancer (CRPC). METHODS: an LC-MS/MS method was developed and validated to quantify GMC1 in plasma and urine from pharmacokinetics studies in rats. An ultra-high-performance liquid chromatography (UHPLC) system equipped with a Waters XTerra MS C18 column was used for chromatographic separation by gradient elution with 0.1% (v/v) formic acid in water and methanol. A Sciex 4000 QTRAP® mass spectrometer was used for analysis by multiple reaction monitoring (MRM) in positive mode; the specific ions [M+H]+m/z 340.995 → m/z 191.000 and [M+H]+ m/z 266.013 → m/z 234.000 were monitored for GMC1 and internal standard (albendazole), respectively. RESULTS: GMC1 and albendazole had retention times of 1.68 and 1.66 min, respectively. The calibration curves for the determination of GMC1 in rat plasma and urine were linear from 1-1000 ng/mL. The LC-MS/MS method was validated with intra- and inter-day accuracy and precision within the 15% acceptance limit. The extraction recovery values of GMC1 from rat plasma and urine were greater than 95.0 ± 2.1% and 97.6 ± 4.6%, respectively, with no significant interfering matrix effect. GMC1 is stable under expected sample handling, storage, preparation and LC-MS/MS analysis conditions. CONCLUSIONS: Pharmacokinetic evaluation of GMC1 revealed that the molecule has a biexponential disposition in rats, is distributed rapidly and extensively, has a long elimination half-life, and appears to be eliminated primarily by first order kinetics.

Management of Hsp90-Dependent Protein Folding by Small Molecules Targeting the Aha1 Co-Chaperone.

Hsp90 plays an important role in health and is a therapeutic target for managing misfolding disease. Compounds that disrupt co-chaperone delivery of clients to Hsp90 target a subset of Hsp90 activities, thereby minimizing the toxicity of pan-Hsp90 inhibitors. Here, we have identified SEW04784 as a first-in-class inhibitor of the Aha1-stimulated Hsp90 ATPase activity without inhibiting basal Hsp90 ATPase. Nuclear magnetic resonance analysis reveals that SEW84 binds to the C-terminal domain of Aha1 to weaken its asymmetric binding to Hsp90. Consistent with this observation, SEW84 blocks Aha1-dependent Hsp90 chaperoning activities, including the in vitro and in vivo refolding of firefly luciferase, and the transcriptional activity of the androgen receptor in cell-based models of prostate cancer and promotes the clearance of phosphorylated tau in cellular and tissue models of neurodegenerative tauopathy. We propose that SEW84 provides a novel lead scaffold for developing therapeutic approaches to treat proteostatic disease.

Functional Comparison of Human and Zebra Fish FKBP52 Confirms the Importance of the Proline-Rich Loop for Regulation of Steroid Hormone Receptor Activity.

Previous studies demonstrated that the 52-kDa FK506-binding protein (FKBP52) proline-rich loop is functionally relevant in the regulation of steroid hormone receptor activity. While zebra fish (Danio rerio; Dr) FKBP52 contains all of the analogous domains and residues previously identified as critical for FKBP52 potentiation of receptor activity, it fails to potentiate activity. Thus, we used a cross-species comparative approach to assess the residues that are functionally critical for FKBP52 function. Random selection of gain-of-function DrFKBP52 mutants in Saccharomyces cerevisiae identified two critical residues, alanine 111 (A111) and threonine 157 (T157), for activation of receptor potentiation by DrFKBP52. In silico homology modeling suggests that alanine to valine substitution at position 111 in DrFKBP52 induces an open conformation of the proline-rich loop surface similar to that observed on human FKBP52, which may allow for sufficient surface area and increased hydrophobicity for interactions within the receptor-chaperone complex. A second mutation in the FKBP12-like domain 2 (FK2), threonine 157 to arginine (T157R), also enhanced potentiation, and the DrFKBP52-A111V/T157R double mutant potentiated receptor activity similar to human FKBP52. Collectively, these results confirm the functional importance of the FKBP52 proline-rich loop, suggest that an open conformation on the proline-rich loop surface is a predictor of activity, and highlight the importance of an additional residue within the FK2 domain.

Nuclear Receptors: A Historical Perspective.

In this chapter, we summarize the birth of the field of nuclear receptors. These receptors exhibit a multitude of roles in cell biology and hence have attracted a great deal of interest in the drug discovery field. It is not certain whether these receptors evolved independently or an ancestral protein acquired various functions upon binding to preexisting small molecules, ligands. Currently, members of this receptor superfamily are categorized in six groups, including "orphan receptors." Research in the area has resulted in several clinically used drugs and continues to reveal further previously unknown roles for these receptors paving the road toward more valuable discoveries in the future.

The Nuclear Receptor Field: A Historical Overview and Future Challenges.

In this article we summarize the birth of the field of nuclear receptors, the discovery of untransformed and transformed isoforms of ligand-binding macromolecules, the discovery of the three-domain structure of the receptors, and the properties of the Hsp90-based heterocomplex responsible for the overall structure of the oligomeric receptor and many aspects of the biological effects. The discovery and properties of the subfamily of receptors called orphan receptors is also outlined. Novel molecular aspects of the mechanism of action of nuclear receptors and challenges to resolve in the near future are discussed.

Evidence for Hsp90 Co-chaperones in Regulating Hsp90 Function and Promoting Client Protein Folding.

Molecular chaperones are a diverse group of highly conserved proteins that transiently interact with partially folded polypeptide chains during normal cellular processes such as protein translation, translocation, and disassembly of protein complexes. Prior to folding or after denaturation, hydrophobic residues that are normally sequestered within a folded protein are exposed to the aqueous environment and are prone to aggregation or misfolding. Multiple classes of molecular chaperones, such as Hsp70s and Hsp40s, recognize and transiently bind polypeptides with exposed hydrophobic stretches in order to prevent misfolding. Other types of chaperones, such as Hsp90, have more specialized functions in that they appear to interact with only a subset of cellular proteins. This chapter focuses on the role of Hsp90 and partner co-chaperones in promoting the folding and activation of a diverse group of proteins with critical roles in cellular signaling and function.

Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52.

Castration-resistant prostate cancer (CRPC) is the emergence of prostate cancer cells that have adapted to the androgen-depleted environment of the prostate. In recent years, targeting multiple chaperones and co-chaperones (e.g., Hsp27, FKBP52) that promote androgen receptor (AR) signaling and/or novel AR regulatory mechanisms have emerged as promising alternative treatments for CRPC. We have shown that inactivation of inhibitor of differentiation 4 (ID4), a dominant-negative helix loop helix protein, promotes de novo steroidogenesis and CRPC with a gene expression signature that resembles constitutive AR activity in castrated mice. In this study, we investigated the underlying mechanism through which loss of ID4 potentiates AR signaling. Proteomic analysis between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(-)ID4) revealed elevated levels of Hsp27 and FKBP52, suggesting a role for these AR-associated co-chaperones in promoting constitutively active AR signaling in L(-)ID4 cells. Interestingly, protein interaction studies demonstrated a direct interaction between ID4 and the 52-kDa FK506-binding protein (FKBP52) in vitro, but not with AR. An increase in FKBP52-dependent AR transcriptional activity was observed in L(-)ID4 cells. Moreover, pharmacological inhibition of FKBP52-AR signaling, by treatment with MJC13, attenuated the tumor growth, weight, and volume in L(-)ID4 xenografts. Together, our results demonstrate that ID4 selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52-mediated AR signaling.

BUILDing SCHOLARS: enhancing diversity among U.S. biomedical researchers in the Southwest.

BACKGROUND AND PURPOSE: With funding from the National Institutes of Health, BUILDing SCHOLARS was established at The University of Texas at El Paso with the goal of implementing, evaluating and sustaining a suite of institutional, faculty and student development interventions in order to train the next generation of biomedical researchers from the U.S. Southwest region, where the need is dire among underserved communities. The focus is on supporting the infrastructure necessary to train and mentor students so they persist on pathways across a range of biomedical research fields. The purpose of this article is to highlight the design and implementation of BUILDing SCHOLARS' key interventions, which offer a systemic student training model for the U.S. Southwest. In-depth reporting of evaluation results is reserved for other technical publications. PROGRAM AND KEY HIGHLIGHTS: BUILDing SCHOLARS uses a comprehensive regional approach to undergraduate training through a multi-institution consortium that includes 12 research partners and various pipeline partners across Texas, New Mexico, and Arizona. Through faculty collaborations and undergraduate research training, the program integrates social and behavioral sciences and biomedical engineering while emphasizing seven transdisciplinary nodes of biomedical research excellence that are common across partner institutions: addiction, cancer, degenerative and chronic diseases, environmental health, health disparities, infectious diseases, and translational biomedicine. Key interventions aim to: (1) improve institutional capacities by expanding undergraduate research training infrastructures; (2) develop an intra- and cross-institutional mentoring-driven "community of practice" to support undergraduate student researchers; (3) broaden the pool of student participants, improve retention, and increase matriculation into competitive graduate programs; and (4) support faculty and postdoctoral personnel by training them in research pedagogy and mentoring techniques and providing them with resources for increasing their research productivity. Student training activities focus on early interventions to maximize retention and on enabling students to overcome common barriers by addressing their educational endowments, science socialization, network development, family expectations, and material resources. Over the long term, BUILDing SCHOLARS will help increase the diversity of the biomedical research workforce in the U.S. by meeting the needs of students from the Southwest region and by serving as a model for other institutions.

The stress regulator FKBP51 drives chronic pain by modulating spinal glucocorticoid signaling.

Polymorphisms in FKBP51 are associated with stress-related psychiatric disorders and influence the severity of pain symptoms experienced after trauma. We report that FKBP51 (FK506 binding protein 51) is crucial for the full development and maintenance of long-term pain states. Indeed, FKBP51 knockout mice, as well as mice in which silencing of FKBP51 is restricted to the spinal cord, showed reduced hypersensitivity in several persistent pain models in rodents. FKBP51 deletion did not compromise the detection of acute painful stimuli, a critical protective mechanism. Moreover, the intrathecal administration of the specific FKBP51 inhibitor SAFit2 reduced the severity of an established pain state, confirming the crucial role of spinal FKBP51 in nociceptive processing. Finally, glucocorticoid signaling, which is known to modulate persistent pain states in rodents, was impaired in FKBP51 knockout mice. This finding suggested that FKBP51 regulates chronic pain by modulation of glucocorticoid signaling. Thus, FKBP51 is a central mediator of chronic pain, likely in humans as well as rodents, and is a new pharmacologically tractable target for the treatment of long-term pain states.

Solution formulation development and efficacy of MJC13 in a preclinical model of castration-resistant prostate cancer.

MJC13, a novel FKBP52 targeting agent, has potential use for the treatment of castration-resistant prostate cancer. The purpose of this work was to develop a solution formulation of MJC13, and obtain its efficacy profile in a human prostate cancer xenograft mouse model. Preformulation studies were conducted to evaluate the physicochemical properties. Co-solvent systems were evaluated for aqueous solubility and tolerance. A human prostate cancer xenograft mouse model was established by growing 22Rv1 prostate cancer cells in C.B-17 SCID mice. The optimal formulation was used to study the efficacy of MJC13 in this preclinical model of castrate-resistant prostate cancer. We found that MJC13 was stable (at least for 1 month), highly lipophilic (logP = 6.49), poorly soluble in water (0.28 µg/mL), and highly plasma protein bound (>98%). The optimal formulation consisting of PEG 400 and Tween 80 (1:1, v/v) allowed us to achieve a MJC13 concentration of 7.5 mg/mL, and tolerated an aqueous environment. After twice weekly intratumoral injection with 10 mg/kg MJC13 in this formulation for four consecutive weeks, tumor volumes were significantly reduced compared to vehicle-treated controls.

Similarities and Distinctions in Actions of Surface-Directed and Classic Androgen Receptor Antagonists.

The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells. These effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared MJC13 and classic AR antagonists such as flutamide and bicalutamide. Microarray analysis and confirmatory qRT-PCR reveals that MJC13 and flutamide inhibit dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells. Both compounds are equally effective on a genome wide basis and as effective as second generation AR antagonists (MDV3100, ARN-509) at selected genes. MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects castrate resistant prostate cancer (CRPC) reveals that it inhibits flutamide activation of an AR mutant (ART877A) that emerges during flutamide withdrawal syndrome, but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. Importantly, MJC13 inhibits AR interactions with SRC2 and ß-catenin in the nucleus and, unlike flutamide, strongly inhibits amplification of AR activity obtained with transfected SRC2 and ß-catenin. MJC13 also inhibits DHT and ß-catenin-enhanced cell division in LNCaP cells. Thus, a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in particular forms of CRPC.

The FKBP52 Cochaperone Acts in Synergy with ß-Catenin to Potentiate Androgen Receptor Signaling.

FKBP52 and ß-catenin have emerged in recent years as attractive targets for prostate cancer treatment. ß-catenin interacts directly with the androgen receptor (AR) and has been characterized as a co-activator of AR-mediated transcription. FKBP52 is a positive regulator of AR in cellular and whole animal models and is required for the development of androgen-dependent tissues. We previously characterized an AR inhibitor termed MJC13 that putatively targets the AR BF3 surface to specifically inhibit FKBP52-regulated AR signaling. Predictive modeling suggests that ß-catenin interacts with the AR hormone binding domain on a surface that overlaps with BF3. Here we demonstrate that FKBP52 and ß-catenin interact directly in vitro and act in concert to promote a synergistic up-regulation of both hormone-independent and -dependent AR signaling. Our data demonstrate that FKBP52 promotes ß-catenin interaction with AR and is required for ß-catenin co-activation of AR activity in prostate cancer cells. MJC13 effectively blocks ß-catenin interaction with the AR LBD and the synergistic up-regulation of AR by FKBP52 and ß-catenin. Our data suggest that co-regulation of AR by FKBP52 and ß-catenin does not require FKBP52 PPIase catalytic activity, nor FKBP52 binding to Hsp90. However, the FKBP52 proline-rich loop that overhangs the PPIase pocket is critical for synergy.

Therapeutic Targeting of the FKBP52 Co-Chaperone in Steroid Hormone Receptor-Regulated Physiology and Disease.

Steroid hormone receptors are ligand-dependent transcription factors that require the dynamic, ordered assembly of multimeric chaperone complexes to reach a functional conformation. Heat shock protein (Hsp) 70 and Hsp90 serve as the central chaperones that mediate this process in conjunction with a variety of co-chaperones. Many of these cochaperones represent potential therapeutic targets for the disruption of Hsp90 client protein function. FKBP52 is an Hsp90-associated co-chaperone that has emerged as a promising therapeutic candidate due to its functional specificity for a small subset of Hsp90 client proteins including androgen (AR), glucocorticoid (GR), and progesterone (PR) receptors. Given its Hsp90-client protein specificity, the targeting of FKBP52 should be more specific and less toxic than the Hsp90- targeting drugs. Additionally, the fkbp52-deficient mice display specific phenotypes related to androgen, progesterone, and glucocorticoid insensitivity suggesting minimal off-target effects. Finally, the fact that FKBP52 is already a validated target of the clinically approved immunosuppressive drug, FK506 (Tacrolimus), indicates that FKBP52 is a "druggable" protein. Thus, the development of FKBP52-specific small molecule inhibitors is predicted to be a highly targeted strategy with potential for the treatment of any disease that is dependent on a functional AR, GR, and/or PR signaling pathway. Much progress has been made in understanding the residues and domains critical for FKBP52 function. The proline-rich loop overhanging the FKBP52 FK1 catalytic domain is functionally important and likely represents an interaction surface within the receptor-chaperone complex. Thus, the targeting of FKBP52 proline-rich loop interactions is the most attractive therapeutic approach to disrupt FKBP52 regulation of receptor activity in steroid hormone receptor-dependent physiology and disease.

Tetratricopeptide repeat domain 9A negatively regulates estrogen receptor alpha activity.

Tetratricopeptide repeat domain 9A (TTC9A) is a target gene of estrogen and progesterone. It is over-expressed in breast cancer. However, little is known about the physiological function of TTC9A. The objectives of this study were to establish a Ttc9a knockout mouse model and to study the consequence of Ttc9a gene inactivation. The Ttc9a targeting vector was generated by replacing the Ttc9a exon 1 with a neomycin cassette. The mice homozygous for Ttc9a exon 1 deletion appear to grow normally and are fertile. However, further characterization of the female mice revealed that Ttc9a deficiency is associated with greater body weight, bigger thymus and better mammary development in post-pubertal mice. Furthermore, Ttc9a deficient mammary gland was more responsive to estrogen treatment with greater mammary ductal lengthening, ductal branching and estrogen target gene induction. Since Ttc9a is induced by estrogen in estrogen target tissues, these results suggest that Ttc9a is a negative regulator of estrogen function through a negative feedback mechanism. This is supported by in vitro evidence that TTC9A over-expression attenuated ERα activity in MCF-7 cells. Although TTC9A does not bind to ERα or its chaperone protein Hsp90 directly, TTC9A strongly interacts with FKBP38 and FKBP51, both of which interact with ERα and Hsp90 and modulate ERα activity. It is plausible therefore that TTC9A negatively regulates ERα activity through interacting with co-chaperone proteins such as FKBP38 and FKBP51.