He, Xiaoping

Abstract Background. We previously reported an alternative ESR1 somatic gain-of-function chromosomal translocation event in a patient presenting with aggressive, endocrine therapy resistant estrogen receptor (ER) positive disease, producing an in-frame fusion gene consisting of N-terminal ESR1 and the C-terminus of the Hippo pathway coactivator YAP1 (ESR1-YAP1). We recently identified another ESR1 fusion through RNA sequencing (RNA-seq) in advanced stage ER+ disease from a chest wall recurrence in a male patient that was refractory to multiple lines of treatment. Two examples of fusions discovered in primary breast cancer samples include ESR1 fused in-frame to C-terminal sequences from NOP2 (ESR1-NOP2), identified in a resistant cohort from a RNA-seq screen focused on 81 primary breast cancers from aromatase inhibitor clinical trials, and a second ESR1 fusion, fused in-frame to the entire coding sequence of POLH (ESR1-POLH), that was identified from RNA-seq analysis of 728 Cancer Genome Atlas breast samples. This current study extends our previous characterization of ESR1-YAP1 by comparing functional and pharmacological properties of these three additional ESR1 gene fusion events of both early stage and advanced breast cancers. Methods. In vitro and in vivo experiments were conducted to test ESR1 fusions to induce therapeutic resistance, and metastasis. The transcriptional and binding properties of each fusion was also examined. Pharmacological inhibition with Palbociclib, a cyclin-dependent kinase 4/6 inhibitor, was utilized to assess drug sensitivity in ESR1 fusion containing breast cancer cells and in a patient derived xenograft (PDX) model expressing ESR1-YAP1 (WHIM18). Results. The YAP1 and PCDH11x fusions conferred estrogen-independent and fulvestrant-resistant growth. Immunohistochemistry revealed significantly higher numbers of ER+ cells in lungs of mice xenografted with T47D cells expressing the YAP1 and PCDH11x fusions compared to YFP control, NOP2 and POLH fusions. Results from ChIP-seq and microarray studies suggest that these two fusions promote proliferation and metastasis through genomic action by binding estrogen response elements (ERE) and subsequent gene activation. We thereby define these fusions as “canonical” fusions compared to “non-canonical” NOP2 and POLH fusions, which demonstrated dramatically decreased genomic binding ability. The non-canonical fusions induced genes associated with basal-like breast cancer and promoted HER2, EGFR, and MAPK gene expression signatures in contrast to genes associated with cell cycle/proliferation induced by canonical fusions. The proliferative ability of canonical fusion-containing ER+ cells was inhibited by Palbociclib in a dose-dependent manner. In vivo WHIM18 tumors in mice fed with Palbociclib-containing chow demonstrated significantly reduced tumor volume, growth rate, and weight compared to tumors in mice on control chow. Conclusions. In-frame ERE activating canonical fusions occur in end-stage drug resistant advanced breast cancer and can be added to ESR1 point mutations as a class of recurrent somatic mutation that may cause acquired resistance. Growth induced by these fusions can be antagonized by Palbociclib and is potentially clinically helpful. Citation Format: Lei JT, Shao J, Zhang J, Iglesia M, Cao J, Chan DW, He X, Kosaka Y, Schmidt C, Matsunuma R, Haricharan S, Crowder R, Hoog J, Phommaly C, Goncalves R, Ramalho S, Lai W-C, Hampton O, Rogers A, Tobias E, Parikh P, Davies S, Ma C, Suman V, Hunt K, Watson M, Hoadley KA, Thompson A, Chen X, Perou CM, Creighton CJ, Maher C, Ellis MJ. Recurrent functionally diverse in-frame ESR1 gene fusions drive endocrine resistance in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD2-03.

Abstract We previously identified serine protease HtrA1 as a down-regulated gene in epithelial ovarian cancer (EOC) that modulates cisplatin-induced cytotoxicity. However, the mechanism by which HtrA1 sensitizes cells to cisplatin-induced cytotoxicity remained unknown. We have shown previously that the pan-cyclin dependent kinase inhibitor flavopiridol (FP) sensitizes cells to cisplatin-induced cytotoxicity; we hypothesized that flavopiridol might therefore modulate the expression of pro-apoptotic proteins. Microarray data indicated that while flavopiridol treatment alone or in combination with cisplatin up-regulated HtrA1 expression by 2-3 fold (providing a means to upregulate HtrA1 expression), treatment with cisplatin alone had no effect. Interestingly, in searching for underlying mechanisms, we noted that WT-1 transcription factor binding elements that is present in HtrA1 promoter is down-regulated by flavopiridol in our microarray studies. Immunoblot analysis revealed that WT-1 is highly expressed in HtrA1-deficient OV167, OV202, PE04 and Ovcar3 cells but not in HtrA1-expressing SKOV3, TOV21G and PE01 cells, suggesting WT-1 negatively regulates HtrA1 expression in EOC. Immunoblot analysis showed FP treatment resulted in down-regulation of WT-1 with a corresponding increase in HtrA1 protein levels in OV202 cells. Additionally, stable down-regulation of WT-1 by short hairpin RNA (shRNA) in OV202 cells increased HtrA1 expression. Conversely, enhanced expression of full length WT-1 isoform in SKOV3 cells downregulated HtrA1 expression. Reporter assay showed that FP treatment upregulated HtrA1 promoter activity in OV202 cells. Reporter assay and chromatin immunoprecipitation experiments showed that WT-1 is specifically recruited to the HtrA1 promoter. Moreover, downregulation of WT-1 expression in OV202 cells increased cisplatin-mediated cytotoxicity as determined by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays – an effect reversed by enhanced expression of HtrA1. Collectively, our studies demonstrate a linkage between the effects of flavopiridol on HtrA1 and its regulatory transcription factor WT-1 that appears in part to explain why flavopiridol has chemosensitizing properties Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4015. doi:10.1158/1538-7445.AM2011-4015

Abstract We have previously identified serine protease HtrA1 as a down-regulated gene in ovarian cancer and loss of HtrA1 may contribute to cell resistance to cisplatin treatment. However, the exact mechanism by which HtrA1 regulates chemosensitivity in ovarian cancer is currently unknown. Here, we used mixture-based oriented peptide library approach and identified XIAP, an important member of the inhibitor of apoptosis (IAP) protein family, as one possible substrate of HtrA1. Recombinant wild type (WT) HtrA1 but not protease-inactive SA mutant HtrA1 can cleave XIAP protein in vitro. Over-expression of HtrA1 led to decreased level of XIAP in OV167 and OV202 ovarian cancer cells, while down-regulation of HtrA1 resulted in increased level of XIAP in SKOV3 ovarian cancer cells. CDDP treatment enhanced the cleavage of XIAP by HtrA1. Immuno-precipitation assay revealed that HtrA1 binds to XIAP directly both in vitro and in vivo. Increased cell resistance to cisplatin treatment was observed in SKOV3 and TOV-21G cells after stable knockdown of HtrA1 with shRNA. In contrast, over-expression of HtrA1 in OV202 cells promotes cell sensitivity to cisplatin treatment which can be reversed by increased expression of XIAP. In conclusion, our experiments reveal XIAP as a novel substrate of HtrA1 and the mechanism by which HtrA1 confers cell sensitivity to cisplatin treatment may be partially through its degradation of XIAP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1531. doi:10.1158/1538-7445.AM2011-1531

Abstract Objectives. To assess chemo-response of patient derived ovarian cancer cells to first line chemotherapeutic agents as well as targeted therapies. Methods. Ascites was obtained at laparotomy from 14 patients with epithelial ovarian cancer. Short term cultures were established in 11 of these and the cells characterized by morphology and immuno-staining to cytokeratin AE1/AE3 and E-cadherin. MTT assays were done on the 11 samples after 48 hours of exposure to Cisplatin, Paclitaxel, Vandetanib and Flavopiridol. Results. Three samples did not grow well and ultimately showed senescence or were over-run by fibroblasts. In the remaining 11 samples, morphology showed the cells to be arranged in a cobble-stone or rosette pattern. Most of the cells stained positive for cytokeratin AE1/AE3 and E-cadherin. The majority of samples (8/11, 73%) were sensitive to either Cisplatin or Paclitaxel. Of the three resistant samples, one was sensitive to Vandetanib, one to Flavopiridol and one was resistant to all four drugs. Two patients did not receive chemotherapy due to morbidity or mortality. Among those who were treated, 4 (50%) had complete response and are currently free of disease. Conclusions. Short term cultures may be a good method of studying chemo-response of tumor cells ex-vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3544.

Abstract Hamartin and tuberin are products of the tumor suppressor genes TSC1 and TSC2, respectively. Mutations affecting either gene result in the tuberous sclerosis syndrome, a neurologic genetic disorder characterized by the formation of multiple benign tumors or hamartomas. In this study, we report the identification of TSC2, but not TSC1, as a substrate of HtrA1, a member of the human HtrA family proteins of serine proteases. We show the direct interaction and colocalization in the cytoplasm of HtrA1 and TSC2 and that HtrA1 cleaves TSC2 both in vitro and in vivo. Finally, we show that alterations in HtrA1 expression cause modifications in phosphorylation status of two downstream targets of TSC2: 4E-BP1 and S6K. Our data suggest that, under particular physiologic or pathologic conditions, HtrA1 degrades TSC2 and activates the downstream targets. Considering that HtrA1 levels are significantly increased during embryogenesis, we speculate that one of the targets of HtrA1 activity during fetal development is the TSC2-TSC1 pathway. Mol Cancer Res; 8(9); 1248–60. ©2010 AACR.

Abstract We previously identified serine protease HtrA1 as a downregulated gene in epithelial ovarian cancer (EOC), but the functional consequence of loss of HtrA1 in EOC remains largely unclear. Here, we report that loss of HtrA1 attenuates anoikis—a critical physiologic barrier for tumor metastasis. In response to loss of anchorage, HtrA1 expression was upregulated in SKOV3 cells, resulting in autocatalytic activation of HtrA1. Stable knockdown of HtrA1 in SKOV3 and TOV21G cells resulted in resistance to anoikis due to enhanced activation of epidermal growth factor receptor (EGFR)/AKT pathway. In suspended SKOV3 cells, enhanced expression of HtrA1 inhibited EGFR/AKT pathway, leading to increased cell death, whereas protease-inactive mutant HtrA1 failed to result in either the inhibition of EGFR/AKT pathway or increased cell death, suggesting the requirement of HtrA1 protease activity in regulating anoikis. Immunoprecipitation and immunofluorescence assays revealed that HtrA1 interacted with EGFR not only on the cell membrane but also in the nucleus. Most importantly, downregulation of HtrA1 significantly enhanced the peritoneal dissemination of SKOV3ip1 cells in nonobese diabetic/severe combined immunodeficient mice, with increased phospho-EGFR level in corresponding tumor nodules compared with that in xenografts originated from the control cells. Taken together, these data reveal for the first time a novel function of HtrA1 in promoting anoikis by attenuating activation of EGFR/AKT pathway that may contribute to its metastasis suppression capacity, thus providing a possible explanation for the aggressive nature of human ovarian tumors with downregulated HtrA1. Cancer Res; 70(8); 3109–18. ©2010 AACR.