Hippo Tumor Suppressor Pathway Research Articles

Analysis of Liver Tumor-Prone Mouse Models of the Hippo Kinase Scaffold Proteins RASSF1A and SAV1.

The tumor suppressor gene RASSF1A is epigenetically silenced in most human cancers. As a binding partner of the kinases MST1 and MST2, the mammalian orthologs of the Drosophila Hippo kinase, RASSF1A is a potential regulator of the Hippo tumor suppressor pathway. RASSF1A shares these properties with the scaffold protein SAV1. The role of this pathway in human cancer has remained enigmatic inasmuch as Hippo pathway components are rarely mutated in tumors. Here we show that Rassf1a homozygous knockout mice develop liver tumors. However, heterozygous deletion of Sav1 or codeletion of Rassf1a and Sav1 produced liver tumors with much higher efficiency than single deletion of Rassf1a. Analysis of RASSF1A-binding partners by mass spectrometry identified the Hippo kinases MST1, MST2, and the oncogenic IκB kinase TBK1 as the most enriched RASSF1A-interacting proteins. The transcriptome of Rassf1a(-/-) livers was more deregulated than that of Sav1(+/-) livers, and the transcriptome of Rassf1a(-/-), Sav1(+/-) livers was similar to that of Rassf1a(-/-) mice. We found that the levels of TBK1 protein were substantially upregulated in livers lacking Rassf1a. Furthermore, transcripts of several β-tubulin isoforms were increased in the Rassf1a-deficient livers presumably reflecting a role of RASSF1A as a microtubule-stabilizing protein. In human liver cancer, RASSF1A frequently undergoes methylation at the promoter but this was not observed for MST1, MST2, or SAV1. Our results suggest a multifactorial role of RASSF1A in suppression of liver carcinogenesis. Cancer Res; 76(9); 2824-35. ©2016 AACR.

Prognostic significance of a component of the Hippo pathway, TAZ, in human uterine endometrioid adenocarcinoma.

Transcriptional coactivator with PDZ-binding motif (TAZ) is a crucial component of the Hippo tumor suppressor pathway, interacting with transcriptional factors to regulate cell proliferation, apoptosis and tumorigenesis. TAZ and its paralog, Yes-associated protein (YAP), are activated at high frequencies during the progression towards malignancy in various tumors. Recently, YAP has been identified to modulate oncogenic features in endometrial adenocarcinoma, and it has also been reported that the nuclear expression of YAP is correlated with the poorly-differentiated form of endometrioid adenocarcinoma. In contrast to YAP, no studies have investigated TAZ expression in endometrioid adenocarcinoma. In the present study, TAZ expression was immunohistochemically examined in 55 clinical samples of endometrioid adenocarcinoma, and the clinical implications were evaluated. The results demonstrated that TAZ was located primarily in the cell nuclei, and that high TAZ expression was significantly correlated with high tumor-factor (P=0.024), stage (P=0.041) and histological grade (P=0.001), lymph node metastasis (P=0.046), recurrence (P=0.002) and a poor prognosis (P=0.007). Furthermore, univariate analysis identified that high TAZ expression was a poor prognostic factor for overall and disease-free survival. To the best of our knowledge, the present case is the first to report the clinical implications of TAZ in endometrioid adenocarcinoma of the uterus. TAZ may become a marker of a poor prognosis in endometrioid adenocarcinoma.

A Potential Structural Switch for Regulating DNA-Binding by TEAD Transcription Factors

TEA domain (TEAD) transcription factors are essential for the normal development of eukaryotes and are the downstream effectors of the Hippo tumor suppressor pathway. Whereas our earlier work established the three-dimensional structure of the highly conserved DNA-binding domain using solution NMR spectroscopy, the structural basis for regulating the DNA-binding activity remains unknown. Here, we present the X-ray crystallographic structure and activity of a TEAD mutant containing a truncated L1 loop, ΔL1 TEAD DBD. Unexpectedly, the three-dimensional structure of the ΔL1 TEAD DBD reveals a helix-swapped homodimer wherein helix 1 is swapped between monomers. Furthermore, each three-helix bundle in the domain-swapped dimer is a structural homolog of MYB-like domains. Our investigations of the DNA-binding activity reveal that although the formation of the three-helix bundle by the ΔL1 TEAD DBD is sufficient for binding to an isolated M-CAT-like DNA element, multimeric forms are deficient for cooperative binding to tandemly duplicated elements, indicating that the L1 loop contributes to the DNA-binding activity of TEAD. These results suggest that switching between monomeric and domain-swapped forms may regulate DNA selectivity of TEAD proteins.

Phosphodiesterase 5 Signals through HIPPO/TAZ Pathway in Maintaining Stemness of Prostate Cancer Cells

Cancer stemness is critical for the metastasis and relapse, however, the mechanism governing the maintenance of cancer stemness remains unknown. Herein, we have investigated the roles of phosphodiesterase 5 (PDE5) in stemness of prostate cancer cells. Both PDE5 and WW domain-containing transcription regulator protein 1 (TAZ), a major co-effector of the Hippo tumor suppressor pathway, are highly expressed in the PC3-derived cancer stem cells (PCSCs), and contribute to maintaining the stemness of PCSCs. Moreover, inhibition of PDE5 activity activates cGMP-dependent protein kinase G (PKG), PKG in turn activates MST/Lats kinases, leads to the cytosolic instability, and attenuates nucleus translocation of TAZ, resulting in inactivation of Hippo signaling. PDE5 inhibitor, vardenafil, not only attenuates the stemness of PCSCs but induces the trans-differentiation of them, these effects are abolished by knockdown of TAZ expression in PCSCs. Finally, in PC3 cells- and PCSCs-derived xenografts, administration of PDE5 inhibitor to the nude mice reverses the stem markers expression, whereas knockdown of TAZ in PC3 cells and PCSCs leads to the decreases in tumorigenesis and elimination of PDE5 inhibitors’ effects. Collectively, the present study demonstrates that inhibition of PDE5 activates hippo signaling to suppress TAZ in maintaining the stemness of PCSCs, and that PDE5 inhibitors could be an effective therapeutic intervention for metastasis and relapse of prostate cancer.

The Hippo/STE20 homolog SIK1 interacts with MOB1 to regulate cell proliferation and cell expansion in Arabidopsis.

Multicellular organisms co-ordinate cell proliferation and cell expansion to maintain organ growth. In animals, the Hippo tumor suppressor pathway is a master regulator of organ size. Central to this pathway is a kinase cascade composed of Hippo and Warts, and their activating partners Salvador and Mob1/Mats. In plants, the Mob1/Mats homolog MOB1A has been characterized as a regulator of cell proliferation and sporogenesis. Nonetheless, no Hippo homologs have been identified. Here we show that the Arabidopsis serine/threonine kinase 1 (SIK1) is a Hippo homolog, and that it interacts with MOB1A to control organ size. SIK1 complements the function of yeast Ste20 in bud site selection and mitotic exit. The sik1 null mutant is dwarf with reduced cell numbers, endoreduplication, and cell expansion. A yeast two-hybrid screen identified Mob1/Mats homologs MOB1A and MOB1B as SIK1-interacting partners. The interaction between SIK1 and MOB1 was found to be mediated by an N-terminal domain of SIK1 and was further confirmed by bimolecular fluorescence complementation. Interestingly, sik1 mob1a is arrested at the seedling stage, and overexpression of neither SIK1 in mob1a nor MOB1A in sik1 can rescue the dwarf phenotypes, suggesting that SIK1 and MOB1 may be components of a larger protein complex. Our results pave the way for constructing a complete Hippo pathway that controls organ growth in higher plants.

The Hippo pathway effectors YAP and TAZ promote cell growth by modulating amino acid signaling to mTORC1.

YAP and TAZ are transcriptional co-activators and function as the major effectors of the Hippo tumor suppressor pathway, which controls cell growth, tissue homeostasis, and organ size. Here we show that YAP/TAZ play an essential role in amino acid-induced mTORC1 activation, particularly under nutrient-limiting conditions. Mechanistically, YAP/TAZ act via the TEAD transcription factors to induce expression of the high-affinity leucine transporter LAT1, which is a heterodimeric complex of SLC7A5 and SLC3A2. Deletion of YAP/TAZ abolishes expression of LAT1 and reduces leucine uptake. Re-expression of SLC7A5 in YAP/TAZ knockout cells restores leucine uptake and mTORC1 activation. Moreover, SLC7A5 knockout cells phenocopies YAP/TAZ knockout cells which exhibit defective mTORC1 activation in response to amino acids. We further demonstrate that YAP/TAZ act through SLC7A5 to provide cells with a competitive growth advantage. Our study provides molecular insight into the mechanism of YAP/TAZ target genes in cell growth regulation.

Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

Metabolic activation is a common feature of many cancer cells and is frequently associated with the clinical outcomes of various cancers, including hepatocellular carcinoma. Thus, aberrantly activated metabolic pathways in cancer cells are attractive targets for cancer therapy. Yes-associated protein 1 (YAP1) and transcriptional coactivator with PDZ-binding motif (TAZ) are oncogenic downstream effectors of the Hippo tumor suppressor pathway, which is frequently inactivated in many cancers. Our study revealed that YAP1/TAZ regulates amino acid metabolism by up-regulating expression of the amino acid transporters solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5). Subsequently, increased uptake of amino acids by the transporters (SLC38A1 and SLC7A5) activates mammalian target of rapamycin complex 1 (mTORC1), a master regulator of cell growth, and stimulates cell proliferation. We also show that high expression of SLC38A1 and SLC7A5 is significantly associated with shorter survival in hepatocellular carcinoma patients. Furthermore, inhibition of the transporters and mTORC1 significantly blocks YAP1/TAZ-mediated tumorigenesis in the liver. These findings elucidate regulatory networks connecting the Hippo pathway to mTORC1 through amino acid metabolism and the mechanism's potential clinical implications for treating hepatocellular carcinoma. YAP1 and TAZ regulate cancer metabolism and mTORC1 through regulation of amino acid transportation, and two amino acid transporters, SLC38A1 and SLC7A5, might be important therapeutic targets.

Overlapping functions of the MAP4K family kinases Hppy and Msn in Hippo signaling.

The Hippo (Hpo) tumor suppressor pathway is an evolutionarily conserved signaling pathway that controls tissue growth and organ size in species ranging from Drosophila to human, and its malfunction has been implicated in many types of human cancer. In this study, we conducted a kinome screen and identified Happyhour (Hppy)/MAP4K3 as a novel player in the Hpo pathway. Our biochemical study showed that Hppy binds and phosphorylates Wts. Our genetic experiments suggest that Hppy acts in parallel and partial redundantly with Misshapen (Msn)/MAP4K4 to regulate Yki nuclear localization and Hpo target gene expression in Drosophila wing imaginal discs. Furthermore, we showed that cytoskeleton stress restricts Yki nuclear localization through Hppy and Msn when Hpo activity is compromised, thus providing an explanation for the Wts-dependent but Hpo-independent regulation of Yki in certain contexts. Our study has unraveled an additional layer of complexity in the Hpo signaling pathway and laid down a foundation for exploring how different upstream regulators feed into the core Hpo pathway.

The interplay between centrosomes and the Hippo tumor suppressor pathway

Centrosome amplification is a common feature of both solid and hematological human malignancies. Extra centrosomes are not merely innocent bystanders in cancer cells, but rather promote tumor progression by disrupting normal cellular architecture and generating chromosome instability. Consequently, centrosome amplification correlates with advanced tumor grade and overall poor clinical prognosis. By contrast, extra centrosomes are adversely tolerated in non-transformed cells and hinder cell proliferation. This suggests that in addition to acquiring extra centrosomes, cancer cells must also adapt to overcome the deleterious consequences associated with them. Here, we review evidence that implicates core components of the Hippo tumor suppressor pathway as having key roles in both the direct and indirect regulation of centrosome number. Intriguingly, functional inactivation of the Hippo pathway, which is common across broad spectrum of human cancers, likely represents one key adaptation that enables cancer cells to tolerate extra centrosomes.

Role of the YAP Oncoprotein in Priming Ras-Driven Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), a cancer characterized by features of skeletal muscle histogenesis, is the most common soft tissue sarcoma of childhood and adolescence. Survival for high-risk groups is less than 30% at 5 years. RMS also occurs during adulthood, with a lower incidence but higher mortality. Recently, mutational profiling has revealed a correlation between activating Ras mutations in the embryonal (eRMS) and pleomorphic (pRMS) histologic variants of RMS, and a poorer outcome for those patients. Independently, the YAP transcriptional coactivator, an oncoprotein kept in check by the Hippo tumor suppressor pathway, is upregulated in eRMS. Here we show that YAP promotes cell proliferation and antagonizes apoptosis and myogenic differentiation of human RMS cells bearing oncogenic Ras mutations in cell culture studies in vitro and in murine xenografts in vivo. Pharmacologic inhibition of YAP by the benzoporphyrin derivative verteporfin decreased cell proliferation and tumor growth in vivo. To interrogate the temporal contribution of YAP in eRMS tumorigenesis, we used a primary human cell-based genetic model of Ras-driven RMS. Constitutively active YAP functioned as an early genetic lesion, permitting bypass of senescence and priming myoblasts to tolerate subsequent expression of hTERT and oncogenic Ras, which were necessary and sufficient to generate murine xenograft tumors mimicking RMS in vivo. This work provides evidence for cooperation between YAP and oncogenic Ras in RMS tumorigenesis, laying the foundation for preclinical co-targeting of these pathways.

Active YAP promotes pancreatic cancer cell motility, invasion and tumorigenesis in a mitotic phosphorylation-dependent manner through LPAR3.

The transcriptional co-activator Yes-associated protein, YAP, is a main effector in the Hippo tumor suppressor pathway. We recently defined a mechanism for positive regulation of YAP through CDK1-mediated mitotic phosphorylation. Here, we show that active YAP promotes pancreatic cancer cell migration, invasion and anchorage-independent growth in a mitotic phosphorylation-dependent manner. Mitotic phosphorylation is essential for YAP-driven tumorigenesis in animals. YAP reduction significantly impairs cell migration and invasion. Immunohistochemistry shows significant upregulation and nuclear localization of YAP in metastases when compared with primary tumors and normal tissue in human. Mitotic phosphorylation of YAP controls a unique transcriptional program in pancreatic cells. Expression profiles reveal LPAR3 (lysophosphatidic acid receptor 3) as a mediator for mitotic phosphorylation-driven pancreatic cell motility and invasion. Together, this work identifies YAP as a novel regulator of pancreatic cancer cell motility, invasion and metastasis, and as a potential therapeutic target for invasive pancreatic cancer.

CDK1 phosphorylation of TAZ in mitosis inhibits its oncogenic activity.

The transcriptional co-activator with PDZ-binding motif (TAZ) is a downstream effector of the Hippo tumor suppressor pathway, which plays important roles in cancer and stem cell biology. Hippo signaling inactivates TAZ through phosphorylation (mainly at S89). In the current study, we define a new layer of regulation of TAZ activity that is critical for its oncogenic function. We found that TAZ is phosphorylated in vitro and in vivo by the mitotic kinase CDK1 at S90, S105, T326, and T346 during the G2/M phase of the cell cycle. Interestingly, mitotic phosphorylation inactivates TAZ oncogenic activity, as the non-phosphorylatable mutant (TAZ-S89A/S90A/S105A/T326A/T346A, TAZ-5A) possesses higher activity in epithelial-mesenchymal transition, anchorage-independent growth, cell migration, and invasion when compared to the TAZ-S89A mutant. Accordingly, TAZ-5A has higher transcriptional activity compared to the TAZ-S89A mutant. Finally, we show that TAZ-S89A or TAZ-5A (to a greater extent) was sufficient to induce spindle and centrosome defects, and chromosome misalignment/missegregation in immortalized epithelial cells. Together, our results reveal a previously unrecognized connection between TAZ oncogenicity and mitotic phospho-regulation.

Abstract POSTER-BIOL-1324A: Loss of DYRK1A promotes transformation of the fallopian tube epithelial cells by oncogenic ras

Abstract Purpose of the study: High-grade serous ovarian carcinoma (HGSOC) is an aggressive form of ovarian cancer that is characterized by nearly ubiquitous presence of somatic mutations in TP53 gene and widespread gene copy number variations. It is likely that some of these genetic alterations cooperate with TP53 mutations in the pathogenesis of HGSOC; however, the specific roles of individual genes that are frequently gained or lost in HGSOC are only beginning to emerge. We have previously characterised an important role of DYRK1A protein kinase in the ability of human cells to undergo G0/G1 cell cycle arrest and oncogenic-Ras induced senescence. DYRK1A is required for the assembly of the DREAM repressor complex that controls the genes involved in cell cycle progression; this function of DYRK1A could be promoted by LATS kinases of the Hippo tumor suppressor pathway. Analysis of the The Cancer Genome Atlas (TCGA) ovarian dataset revealed loss of heterozygosity (LOH) of DYRK1A gene in over 25% HGSOC tumors. Therefore, we sought to determine whether loss of DYRK1A function can contribute to ovarian carcinogenesis. Experimental Procedures: A panel of human ovarian cancer cell lines was used to determine the levels of DYRK1A and its ability to suppress cell proliferation. The fallopian tube epithelial cell (FTEC) cell lines with stable shRNA knock-down of DYRK1A were used in transformation assays with ovarian cancer-relevant oncogenes. Bioinformatics analysis of the TGCA ovarian oncogenomic dataset was applied to determine whether DYRK1A loss in HGSOC could lead to perturbation of any specific gene expression programs. Summary of Data: It was found that depletion of DYRK1A promotes oncogenic transformation of the FTEC cells expressing mutant p53R175H and oncogenic HRASG12V. Rescue of DYRK1A expression in these cells resulted in arrest of proliferation and senescence. DYRK1A protein levels were reduced in a subset of ovarian cancer cell lines; overexpression of active but not the kinase-inactive DYRK1A suppressed the proliferation of these cells. Gene Set Enrichment Analysis (GSEA) of the TCGA HGSOC dataset revealed upregulated expression of the DREAM target genes in a subset of samples with LOH of both DYRK1A and LATS1 genes. Conclusion: Our findings support the role of DYRK1A as an ovarian tumor supressor and suggest that loss of DYRK1A function in HGSOC could lead to activation of transcription of the genes involved in cell proliferation. Citation Format: Larisa Litovchick, Siddharth Saini, Alison Karst, Lan Hu, Ronny Drapkin, James A. DeCaprio. Loss of DYRK1A promotes transformation of the fallopian tube epithelial cells by oncogenic ras [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1324A.

Abstract 4956: Hsp27 negatively affects Hippo tumor suppressor pathway to regulate cell survival in cancer

Abstract Introduction: Heat shock protein 27 (Hsp27) is a molecular chaperone highly and uniformly expressed in treatment resistant cancers like castrate resistant prostate cancer (CRPC). Hsp27 regulates activity of several oncogenic pathways and its levels correlate with aggressive tumor behaviour, drug resistance and tumor growth. Similarly, dysregulation of the Hippo tumor suppressor pathway, which restricts organ size and cell proliferation, occurs in many types of cancers. In healthy cells, activation of the Hippo pathway results in phosphorylation and cytoplasmic retention of two transcriptional co-activators YAP and TAZ, whereas in cancer YAP/TAZ are free to translocate to the nucleus and increase cell proliferation by promoting the activities of certain transcriptional factors including TEAD1. Inactivation of the Hippo pathway correlates with poor patient outcome and progression of tumors as well as an increase in migration, invasion and metastatic potential of cancer cells. Therefore it is of great importance to establish the correlation between Hsp27 and the Hippo pathway to further discover suitable targets in the treatment of metastatic malignancies. Methods: Hsp27 gain and loss of function experiments were done on 3 different cancer cell lines and the functional effects on every step of the pathway were monitored via Western blots and Immunofluorescence. Activity of YAP/TAZ after Hsp27 gain and loss of function was monitored by conducting qRT-PCR on TEAD target genes. Transcriptional activity of TEAD1 was also examined using a TEAD-dependent Luciferase reporter construct. Co-immunoprecipitation assay was conducted to analyze protein interactions in the absence/presence of Hsp27. Pathway activity in prostate cancer will be assessed by immunohistochemistry staining of core components of the pathway in patients’ tissue samples. Results: Our preliminary findings indicate that Hsp27 negatively affects the Hippo pathway. We found that targeting Hsp27 using siRNA in the PC3 (prostate cancer), A549 (lung cancer) and MDA-MB-453 (triple negative breast cancer) leads to increased cytoplasmic retention of p-YAP compared to control siRNA treated cells. Moreover, using immunofluorescence, we observed reduced nuclear translocation of the YAP/TAZ as well as sequestration of these components with cytoplasmic 14-3-3 proteins in siRNA treated PC3 cells. Furthermore inhibition of Hsp27 in prostate and lung tumour cells resulted in suppression of TEAD transcriptional activity analyzed by qRT-PCR and luciferase assay. Hsp27 over-expression experiments yielded opposite results compared to knockdown. Conclusion: Hsp27 overexpression contributes to inactivation of Hippo pathway. Targeting Hsp27 leads to inactivation of YAP and TAZ onco-proteins affecting cancer cell survival. Impact: Our data further supports the significance of targeting Hsp27 as a treatment option in cancers, especially metastatic malignancies like CRPC. Citation Format: Sepideh Vahid, Daksh Thaper, Amina Zoubeidi. Hsp27 negatively affects Hippo tumor suppressor pathway to regulate cell survival in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4956. doi:10.1158/1538-7445.AM2015-4956

Abstract LB-200: Loss of the scaffold protein Kibra in a mouse model of triple-negative breast cancer

Abstract Introduction: Targeted therapies in breast cancer rely on tumor cell expression of Estrogen and/or HER2 receptors. Triple negative breast cancers (TNBCs), lacking these receptors, have no targeted therapies. We have developed a preclinical murine model expressing a naturally occurring oncogenic variant of the Met receptor in the mammary gland combined with loss of function of the tumour suppressor p53 (MMTV-Met;Trp53fl/+;Cre). This model recapitulates many features of TNBC at the level of gene expression, pathological markers and genomic alterations. Notably, this model spontaneously undergoes loss of a genomic region syntenic with human chromosome 5q which is lost in up to 70% of human TNBCs (Turner et al., 2010). This provides an opportunity for us to identify and study potential driver genes for this breast cancer subtype. Experimental procedures: Approximately 80% of MMTV-Met;Trp53fl/+;Cre mammary tumors have a mesenchymal pathology that recapitulates key features of TNBC. Array CGH profiling showed a consistent loss of chromosome 11:31.35-58.2Mb, syntenic with human 5q31.1 and 5q33.1-35.3. Analysis of mouse model expression profiling data confirmed decreased expression of 83 genes within this region. Comparison with human breast cancers within the TCGA dataset further highlighted 14 of these genes with significantly decreased expression in humans with the TNBC subtypes ‘Basal’ and ‘Claudin-low’. One of these genes, WWC1, undergoes heterozygous deletion in 49% of human Basal breast cancers (TCGA, Nature 2012) and has also been associated with Claudin-low cancers (Moleirinho et al., 2013). WWC1, also known as Kibra, encodes a scaffold protein that positively regulates the Hippo tumor suppressor pathway. We used both Kibra knockdown and over-expression to understand the functional consequences of Kibra-loss. Results: MMTV-Met solid carcinoma cells retain both Kibra expression and epithelial characteristics typical of human luminal breast cancers (Ponzo et al., 2009). Kibra depletion by siRNA in these cells led to weakening of cell-cell junctions and colony dispersal, as well as decreased cortical actin and increased membrane protrusions, consistent with epithelial-to-mesenchymal transition (EMT). Mice injected with Kibra shRNA-tumor cells developed secondary lesions at sites distant to the mammary gland. By comparison, re-expression of Kibra in MMTV-Met;Trp53fl/+ cell lines and human TNBC cells led to morphological changes consistent with an EMT reversal, including loss of actin rich protrusions. Furthermore, re-expressing Kibra impaired proliferation in vitro and decreased tumorigenicity in vivo. Globally, our results strongly suggest that Kibra loss plays a significant role in TNBC development and metastasis and is a candidate driver gene for Chr5q loss. Citation Format: Jennifer France Knight, Tina Gruosso, Danielle Angeline de Verteuil, Sadiq Saleh, Robert Lesurf, Hong Zhao, Ryan Davis, Dongmei Zuo, Robert Cardiff, Jeffrey Gregg, Michael Hallett, Morag Park. Loss of the scaffold protein Kibra in a mouse model of triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-200. doi:10.1158/1538-7445.AM2015-LB-200

Abstract 1956: A subset of prostate cancer cells with loss of MST1 expression confers resistance to castration through YAP1-AR interactions

Abstract MST1 is a core kinase component of the Hippo tumor suppressor pathway. YAP1 is a transcriptional cofactor and a prominent nuclear effector of the Hippo signaling. YAP1 activation is linked to cancer promotion with poor prognosis. However, the role of MST1-YAP axis in prostate cancer (PC) progression remains unknown. Here we isolated MST1 high and MST1 low/- expressing cell subsets from a well-characterized LNCaP PC cell model by employing novel method that utilizes an epigenetically regulated-MST1 promoter-driven GFP reporter combined with FACS. We performed molecular and functional characterizations of these cells utilizing a wide variety of experimental approaches. We demonstrated that unlike MST1 high, MST1 low/- cells shared the features of cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) phenotype, showed superior osteogenic abilities, and were resistant to androgen depletion and enzalutamide (ENZ), a potent inhibitor of androgen receptor (AR). Consistently, silencing of MST1 expression by siRNA recapitulated the above molecular and function behaviors of MST1 low/- cells. In addition, MST1 low/- cells showed superior invasive growth abilities in vitro and tumor-forming abilities with metastatic potentials in mice compared with those of MST1 high cells. Moreover, relative YAP1 expression and its nuclear localization were much higher in MST1 low/- cells than MST1 high cells. Likewise, YAP1 protein levels and its nuclear abundance were increased in PC clinical samples compared to non-cancerous counters. We further showed that YAP1 interacted with AR in an androgen-dependent manner in LNCaP cells, whereas the interactions between the two proteins were androgen-independent (AI) in C4-2 CRPC cells. Furthermore we found that unlike LNCaP, YAP1-AR interactions were resistant to ENZ exposure in C4-2 cells. Consistently, silencing of MST1 in LNCaP cells augmented AI interactions between YAP1 and AR proteins. Finally, the disruption of YAP1 signaling by RNAi and Verteporfin (VP) attenuated C4-2 cell growth and promoted apoptosis, accompanied with the attenuation of AR-dependent gene expression. In conclusion, our findings suggest that the Hippo-YAP1-AR signaling axis is one of the key mechanisms for metastatic PC progression and may represent as a promising therapeutic target for patients with the lethal disease. Note: This abstract was not presented at the meeting. Citation Format: Bekir Cinar, Gamze Kuser Abali, Michael Lewis, Colm Morrissey, Isla Garraway. A subset of prostate cancer cells with loss of MST1 expression confers resistance to castration through YAP1-AR interactions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1956. doi:10.1158/1538-7445.AM2015-1956

Abstract 1965: Hippo pathway restrains cell proliferation via assembly of the DREAM repressor complex

Abstract Hippo pathway mediates tumor suppression in various organisms by the mechanisms that are not fully understood. The canonical Hippo pathway converges on the pro-oncogenic transcription factors YAP and TAZ that are phosphorylated by LATS1 or LATS2 protein kinases, resulting in their proteasomal degradation. Hippo pathway is regulated by various extracellular signals including lysophosphatidic acid (LPA), a bioactive lipid that has a tumor-promoting activity. High levels of LPA in the malignant effusions correlate with increased epithelial ovarian cancer (EOC) aggressiveness. However, the mitogenic effect of LPA is not well understood. We hypothesized that the Hippo signaling can regulate cell proliferation though a novel mechanism involving retinoblastoma (RB) family proteins. Previous studies show that LATS2 contributes to the function of the DREAM (DP, RB-like, E2F, and MuvB core) complex that promotes the G0/G1 arrest by repressing more than 800 cell cycle-regulated genes. The DREAM complex formation depends on phosphorylation of LIN52 subunit of the MuvB core by DYRK1A protein kinase. DYRK1A activity towards LIN52 is promoted by LATS2 phosphorylation in vitro. However, it remains to be established whether the Hippo signaling regulates the function of DREAM complex in vivo. To understand the mechanism by which inhibition of the Hippo signaling can promote cell proliferation, we determined the effect of LPA on the DREAM complex in the ovarian cancer and the precursor cell lines. Using immunoprecipitation-Western blot assays, we show that the LPA treatment of serum-starving cells prevents the recruitment of RB-like p130 into the DREAM complex. Consequently, the cells escape from G0/G1 arrest as demonstrated by FACS analysis of the DNA synthesis. Since MuvB core also contributes to the expression of mitotic genes through binding to BMYB transcription factor, we show that this interaction is not disrupted by LPA. In support of the model that LPA effects are mediated by inhibition of the Hippo signaling, the RNAi knockdown of both LATS1 and LATS2 in ovarian cell lines also resulted in the impaired DREAM assembly. To characterize the mechanism by which LPA can interfere with the DREAM complex formation, we investigated the changes in LIN52 phosphorylation upon LPA treatment or LATS depletion. Our data support the model in which the disruption of the Hippo signaling inhibits the phosphorylation of LIN52, thereby preventing the formation of the DREAM complex and entry into G0/G1 arrest. Our findings highlight a novel mechanism by which inhibition of the Hippo tumor suppressor pathway can promote cancer cell proliferation. Citation Format: Larisa Litovchick, Siddharth Saini. Hippo pathway restrains cell proliferation via assembly of the DREAM repressor complex. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1965. doi:10.1158/1538-7445.AM2015-1965

Mechanism of action of a WWTR1(TAZ)-CAMTA1 fusion oncoprotein

The WWTR1 (protein is known as TAZ)-CAMTA1 (WC) fusion gene defines epithelioid hemangioendothelioma, a malignant vascular cancer. TAZ (transcriptional coactivator with PDZ binding motif) is a transcriptional coactivator and end effector of the Hippo tumor suppressor pathway. It is inhibited by phosphorylation by the Hippo kinases LATS1 and LATS2. Such phosphorylation causes cytoplasmic localization, 14-3-3 protein binding and the phorphorylation of a terminal phosphodegron promotes ubiquitin-dependent degradation (the phosphorylation of the different motifs has several effects). CAMTA1 is a putative tumor suppressive transcription factor. Here we demonstrate that TAZ-CAMTA1 (TC) fusion results in its nuclear localization and constitutive activation. Consequently, cells expressing TC display a TAZ-like transcriptional program that causes resistance to anoikis and oncogenic transformation. Our findings elucidate the mechanistic basis of TC oncogenic properties, highlight that TC is an important model to understand how the Hippo pathway can be inhibited in cancer, and provide approaches for targeting this chimeric protein.

Estrogen regulates Hippo signaling via GPER in breast cancer.

The G protein-coupled estrogen receptor (GPER) mediates both the genomic and nongenomic effects of estrogen and has been implicated in breast cancer development. Here, we compared GPER expression in cancerous tissue and adjacent normal tissue in patients with invasive ductal carcinoma (IDC) of the breast and determined that GPER is highly upregulated in cancerous cells. Additionally, our studies revealed that GPER stimulation activates yes-associated protein 1 (YAP) and transcriptional coactivator with a PDZ-binding domain (TAZ), 2 homologous transcription coactivators and key effectors of the Hippo tumor suppressor pathway, via the Gαq-11, PLCβ/PKC, and Rho/ROCK signaling pathways. TAZ was required for GPER-induced gene transcription, breast cancer cell proliferation and migration, and tumor growth. Moreover, TAZ expression positively correlated with GPER expression in human IDC specimens. Together, our results suggest that the Hippo/YAP/TAZ pathway is a key downstream signaling branch of GPER and plays a critical role in breast tumorigenesis.

A novel role for microRNA-129-5p in inhibiting ovarian cancer cell proliferation and survival via direct suppression of transcriptional co-activators YAP and TAZ.

Transcriptional co-activator Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are key oncogenes in mammalian cells. Activities of YAP and TAZ are largely restricted by the Hippo tumor suppressor pathway through phosphorylation-ubiquitination mechanisms. The involvement of microRNA in cancer progression has recently been reported, though whether they have a role in activating YAP and TAZ in human cancer cells remains unclear. Here, we report a microRNA, miR-129-5p, directly represses YAP and TAZ expression, leading to the inactivation of TEA domain (TEAD) transcription, and the downregulation of Hippo downstream genes, connective tissue growth factor (CTGF) and Cyclin A. Furthermore, we reveal miR-129-5p inhibits ovarian cancer cell proliferation, survival and tumorigenicity, and that downregulation of miR-129-5p in ovarian cancer cells highly correlates with malignant progression and poor survival. Hence, we demonstrate a novel mechanism for YAP and TAZ activation in cancers, indicating not only a potentially pivotal role for miR-129-5p in the progression of ovarian cancer, but also offering new therapeutic strategies to circumvent the disease.