Hanna Y. Irie

The Proteomic Landscape of Triple-Negative Breast Cancer

Triple-negative breast cancer is a heterogeneous disease characterized by poor clinical outcomes and a shortage of targeted treatment options. To discover molecular features of triple-negative breast cancer, we performed quantitative proteomics analysis of twenty human-derived breast cell lines and four primary breast tumors to a depth of more than 12,000 distinct proteins. We used this data to identify breast cancer subtypes at the protein level and demonstrate the precise quantification of biomarkers, signaling proteins, and biological pathways by mass spectrometry. We integrated proteomics data with exome sequence resources to identify genomic aberrations that affect protein expression. We performed a high-throughput drug screen to identify protein markers of drug sensitivity and understand the mechanisms of drug resistance. The genome and proteome provide complementary information that, when combined, yield a powerful engine for therapeutic discovery. This resource is available to the cancer research community to catalyze further analysis and investigation.

PTK6 inhibition promotes apoptosis of Lapatinib-resistant Her2+ breast cancer cells by inducing Bim

IntroductionProtein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase that is highly expressed in Human Epidermal Growth Factor 2+ (Her2+) breast cancers. Overexpression of PTK6 enhances anchorage-independent survival, proliferation, and migration of breast cancer cells. We hypothesized that PTK6 inhibition is an effective strategy to inhibit growth and survival of Her2+ breast cancer cells, including those that are relatively resistant to Lapatinib, a targeted therapy for Her2+ breast cancer, either intrinsically or acquired after continuous drug exposure.MethodsTo determine the effects of PTK6 inhibition on Lapatinib-resistant Her2+ breast cancer cell lines (UACC893R1 and MDA-MB-453), we used short hairpin ribonucleic acid (shRNA) vectors to downregulate PTK6 expression. We determined the effects of PTK6 downregulation on growth and survival in vitro and in vivo, as well as the mechanisms responsible for these effects.ResultsLapatinib treatment of “sensitive” Her2+ cells induces apoptotic cell death and enhances transcript and protein levels of Bim, a pro-apoptotic Bcl2 family member. In contrast, treatment of relatively “resistant” Her2+ cells fails to induce Bim or enhance levels of cleaved, poly-ADP ribose polymerase (PARP). Downregulation of PTK6 expression in these “resistant” cells enhances Bim expression, resulting in apoptotic cell death. PTK6 downregulation impairs growth of these cells in in vitro 3-D MatrigelTM cultures, and also inhibits growth of Her2+ primary tumor xenografts. Bim expression is critical for apoptosis induced by PTK6 downregulation, as co-expression of Bim shRNA rescued these cells from PTK6 shRNA-induced death. The regulation of Bim by PTK6 is not via changes in Erk/MAPK or Akt signaling, two pathways known to regulate Bim expression. Rather, PTK6 downregulation activates p38, and pharmacological inhibition of p38 activity prevents PTK6 shRNA-induced Bim expression and partially rescues cells from apoptosis.ConclusionsPTK6 downregulation induces apoptosis of Lapatinib-resistant Her2+ breast cancer cells by enhancing Bim expression via p38 activation. As Bim expression is a critical biomarker for response to many targeted therapies, PTK6 inhibition may offer a therapeutic approach to treating patients with Her2 targeted therapy-resistant breast cancers.

Inferred miRNA activity identifies miRNA-mediated regulatory networks underlying multiple cancers

MOTIVATION MicroRNAs (miRNAs) play a key role in regulating tumor progression and metastasis. Identifying key miRNAs, defined by their functional activities, can provide a deeper understanding of biology of miRNAs in cancer. However, miRNA expression level cannot accurately reflect miRNA activity. RESULTS We developed a computational approach, ActMiR, for identifying active miRNAs and miRNA-mediated regulatory mechanisms. Applying ActMiR to four cancer datasets in The Cancer Genome Atlas (TCGA), we showed that (i) miRNA activity was tumor subtype specific; (ii) genes correlated with inferred miRNA activities were more likely to enrich for miRNA binding motifs; (iii) expression levels of these genes and inferred miRNA activities were more likely to be negatively correlated. For the four cancer types in TCGA we identified 77-229 key miRNAs for each cancer subtype and annotated their biological functions. The miRNA-target pairs, predicted by our ActMiR algorithm but not by correlation of miRNA expression levels, were experimentally validated. The functional activities of key miRNAs were further demonstrated to be associated with clinical outcomes for other cancer types using independent datasets. For ER(-)/HER2(-) breast cancers, we identified activities of key miRNAs let-7d and miR-18a as potential prognostic markers and validated them in two independent ER(-)/HER2(-) breast cancer datasets. Our work provides a novel scheme to facilitate our understanding of miRNA. In summary, inferred activity of key miRNA provided a functional link to its mediated regulatory network, and can be used to robustly predict patients survival. AVAILABILITY AND IMPLEMENTATION the software is freely available at http://research.mssm.edu/integrative-network-biology/Software.html. CONTACT jun.zhu@mssm.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

PTK6 Inhibition Suppresses Metastases of Triple-Negative Breast Cancer via SNAIL-Dependent E-Cadherin Regulation

Patients with triple-negative breast cancers (TNBC) are at high risk for recurrent or metastatic disease despite standard treatment, underscoring the need for novel therapeutic targets and strategies. Here we report that protein tyrosine kinase 6 (PTK6) is expressed in approximately 70% of TNBCs where it acts to promote survival and metastatic lung colonization. PTK6 downregulation in mesenchymal TNBC cells suppressed migration and three-dimensional culture growth, and enhanced anoikis, resistance to which is considered a prerequisite for metastasis. PTK6 downregulation restored E-cadherin levels via proteasome-dependent degradation of the E-cadherin repressor SNAIL. Beyond being functionally required in TNBC cells, kinase-active PTK6 also suppressed E-cadherin expression, promoted cell migration, and increased levels of mesenchymal markers in nontransformed MCF10A breast epithelial cells, consistent with a role in promoting an epithelial-mesenchymal transition (EMT). SNAIL downregulation and E-cadherin upregulation mediated by PTK6 inhibition induced anoikis, leading to impaired metastatic lung colonization in vivo Finally, effects of PTK6 downregulation were phenocopied by treatment with a recently developed PTK6 kinase inhibitor, further implicating kinase activity in regulation of EMT and metastases. Our findings illustrate the clinical potential for PTK6 inhibition to improve treatment of patients with high-risk TNBC. Cancer Res; 76(15); 4406-17. ©2016 AACR.

EPRS is a critical regulator of cell proliferation and estrogen signaling in ER + breast cancer

Aminoacyl tRNA synthetases (ARSs) are a class of enzymes with well-conserved housekeeping functions in cellular translation. Recent evidence suggests that ARS genes may participate in a wide array of cellular processes, and may contribute to the pathology of autoimmune disease, cancer, and other diseases. Several studies have suggested a role for the glutamyl prolyl tRNA synthetase (EPRS) in breast cancers, although none has identified any underlying mechanism about how EPRS contributes to carcinogenesis. In this study, we identified EPRS as upregulated in estrogen receptor positive (ER+) human breast tumors in the TCGA and METABRIC cohorts, with copy number gains in nearly 50% of samples in both datasets. EPRS expression is associated with reduced overall survival in patients with ER+ tumors in TCGA and METABRIC datasets. EPRS expression was also associated with reduced distant relapse-free survival in patients treated with adjuvant tamoxifen monotherapy for five years, and EPRS-correlated genes were highly enriched for genes predictive of a poor response to tamoxifen. We demonstrated the necessity of EPRS for proliferation of tamoxifen-resistant ER+ breast cancer, but not ER- breast cancer cells. Transcriptomic profiling showed that EPRS regulated cell cycle and estrogen response genes. Finally, we constructed a causal gene network based on over 2500 ER+ breast tumor samples to build up an EPRS-estrogen signaling pathway. EPRS and its regulated estrogenic gene network may offer a promising alternative approach to target ER+ breast cancers that are refractory to current anti-estrogens.

PRKCQ promotes oncogenic growth and anoikis resistance of a subset of triple- negative breast cancer cells

BackgroundThe protein kinase C (PKC) family comprises distinct classes of proteins, many of which are implicated in diverse cellular functions. Protein tyrosine kinase C theta isoform (PRKCQ)/PKCθ, a member of the novel PKC family, may have a distinct isoform-specific role in breast cancer. PKCθ is preferentially expressed in triple-negative breast cancer (TNBC) compared to other breast tumor subtypes. We hypothesized that PRKCQ/PKCθ critically regulates growth and survival of a subset of TNBC cells.MethodsTo elucidate the role of PRKCQ/PKCθ in regulating growth and anoikis resistance, we used both gain and loss of function to modulate expression of PRKCQ. We enhanced expression of PKCθ (kinase-active or inactive) in non-transformed breast epithelial cells (MCF-10A) and assessed effects on epidermal growth factor (EGF)-independent growth, anoikis, and migration. We downregulated expression of PKCθ in TNBC cells, and determined effects on in vitro and in vivo growth and survival. TNBC cells were also treated with a small molecule inhibitor to assess requirement for PKCθ kinase activity in the growth of TNBC cells.ResultsPRKCQ/PKCθ can promote oncogenic phenotypes when expressed in non-transformed MCF-10A mammary epithelial cells; PRKCQ/PKCθ enhances anchorage-independent survival, growth-factor-independent proliferation, and migration. PKCθ expression promotes retinoblastoma (Rb) phosphorylation and cell-cycle progression under growth factor-deprived conditions that typically induce cell-cycle arrest of MCF-10A breast epithelial cells. Proliferation and Rb phosphorylation are dependent on PKCθ-stimulated extracellular signal-related kinase (Erk)/mitogen-activated protein kinase (MAPK) activity. Enhanced Erk/MAPK activity is dependent on the kinase activity of PKCθ, as overexpression of kinase-inactive PKCθ does not stimulate Erk/MAPK or Rb phosphorylation or promote growth-factor-independent proliferation. Downregulation of PRKCQ/PKCθ in TNBC cells enhances anoikis, inhibits growth in 3-D MatrigelTM cultures, and impairs triple-negative tumor xenograft growth. AEB071, an inhibitor of PKCθ kinase activity, also inhibits growth and invasive branching of TNBC cells in 3-D cultures, further supporting a role for PKCθ kinase activity in triple-negative cancer cell growth.ConclusionsEnhanced PRKCQ/PKCθ expression can promote growth-factor-independent growth, anoikis resistance, and migration. PRKCQ critically regulates growth and survival of a subset of TNBC. Inhibition of PKCθ kinase activity may be an attractive therapeutic approach for TNBC, a subtype in need of improved targeted therapies.

EMUDRA: Ensemble of Multiple Drug Repositioning Approaches to improve prediction accuracy

Motivation Availability of large-scale genomic, epigenetic and proteomic data in complex diseases makes it possible to objectively and comprehensively identify the therapeutic targets that can lead to new therapies. The Connectivity Map has been widely used to explore novel indications of existing drugs. However, the prediction accuracy of the existing methods, such as Kolmogorov-Smirnov statistic remains low. Here we present a novel high-performance drug repositioning approach that improves over the state-of-the-art methods. Results We first designed an expression weighted cosine (EWCos) method to minimize the influence of the uninformative expression changes and then developed an ensemble approach termed ensemble of multiple drug repositioning approaches (EMUDRA) to integrate EWCos and three existing state-of-the-art methods. EMUDRA significantly outperformed individual drug repositioning methods when applied to simulated and independent evaluation datasets. We predicted using EMUDRA and experimentally validated an antibiotic rifabutin as an inhibitor of cell growth in triple negative breast cancer. EMUDRA can identify drugs that more effectively target disease gene signatures and will thus be a useful tool for identifying novel therapies for complex diseases and predicting new indications for existing drugs. Availability and implementation The EMUDRA R package is available at doi: 10.7303/syn11510888. Supplementary information Supplementary data are available at Bioinformatics online.

PTK6 regulates growth and survival of endocrine therapy-resistant ER+ breast cancer cells

AbstractThe non-receptor tyrosine kinase, PTK6/BRK, is highly expressed in multiple tumor types, including prostate, ovarian, and breast cancers, and regulates oncogenic phenotypes such as proliferation, migration, and survival. PTK6 inhibition also overcomes targeted therapy resistance of HER2+ breast cancer. Although PTK6 is highly expressed in ER+ Luminal breast cancers, the role of PTK6 in this subtype has not been elucidated. In this study, we investigated the functions of PTK6 in ER+ Luminal breast cancer cells, including those that are relatively resistant to estrogen deprivation or targeted endocrine therapies used in the treatment of ER+ cancers. Enhanced expression of PTK6 in ER+ breast cancer cells enhances growth of ER+ breast cancer cells, including tamoxifen-treated cells. Downregulation of PTK6 in ER+ breast cancer cells, including those resistant to tamoxifen, fulvestrant, and estrogen deprivation, induces apoptosis, as evidenced by increased levels of cleaved PARP, and an increase in the AnnexinV+ population. PTK6 downregulation impairs growth of these cells in 3D MatrigelTM cultures, and virtually abrogates primary tumor growth of both tamoxifen-sensitive and resistant MCF-7 xenografts. Finally, we show that p38 MAPK activation is critical for PTK6 downregulation-induced apoptosis, a mechanism that we previously reported for survival of HER2+ breast cancer cells, highlighting conserved mechanisms of survival regulation by PTK6 across breast cancer subtypes. In conclusion, our studies elucidate critical functions of PTK6 in ER+ Luminal breast cancers and support PTK6 as an attractive therapeutic target for ER+ breast cancers.Therapeutics: enzyme provides drug target for ER+ breast cancerDrugs that target a tumor-promoting enzyme called protein tyrosine kinase 6 (PTK6) could help treat hormone-receptor positive breast cancer. A team led by Hanna Irie from the Icahn School of Medicine at Mount Sinai in New York, NY, USA, investigated the role of PTK6, also known as breast tumor kinase, in breast cancer cells that grow in response to the hormone estrogen. They boosted the enzyme’s expression in estrogen receptor-expressing breast cancer cells and saw enhanced growth. Conversely, downregulating PTK6 levels led to cell death, including in tumor cells that were resistant to tamoxifen
 and other therapies commonly used to treat estrogen-receptor positive breast cancer. The researchers showed that this effect was dependent on the activation of a signaling pathway previously found to be important in another subtype of breast cancer, highlighting a conserved mechanism of cell survival regulation by PTK6.

Abstract B22: Novel dual regulation of epithelial-mesenchymal transition and anoikis resistance prevents metastases of triple negative breast cancer

Triple negative breast cancers (TNBC) are associated with a higher rate of metastatic recurrence and poorer prognosis due to intrinsic biological aggressiveness and lack of targeted therapies. Novel strategies to prevent metastasis are needed to improve outcomes for patients diagnosed with TNBC. Epithelial-mesenchymal transition (EMT) and anoikis resistance are processes recognized as contributing to treatment resistance and enhanced metastatic potential. In a functional genomic screen, we identified several candidates as novel dual regulators of EMT and anoikis sensitivity of triple negative breast cancer cells, that can be pharmacologically targeted to prevent metastases. One highly validated candidate, PTK6/Brk, specifically enhances anoikis resistance and metastatic potential of triple negative breast cancer cells via E-cadherin regulation. Overexpression of PTK6 in non-transformed immortalized breast epithelial cells is sufficient to promote an EMT, whereas PTK6 inhibition (either PTK6 shRNA or kinase inhibitor treatment) enhances E-cadherin expression and suppresses migration and lung colonization of TNBC cells. PTK6-dependent E-cadherin regulation and anoikis resistance is specifically dependent on levels of SNAIL, a transcriptional repressor. SNAIL down-regulation by PTK6 inhibition is directly responsible for modulation of anoikis sensitivity, which is in turn causally linked to lung colonization potential. PTK6 inhibition promotes the degradation of SNAIL via a novel mechanism independent of GSK3β and Fbox proteins known to regulate Snail ubiquitination. Using an siRNA screening approach, we identified novel E3 ligase candidates responsible for SNAIL ubiquitination and degradation downstream of PTK6 inhibition. PTK6 is a representative novel regulator of EMT and anoikis resistance that can be targeted to prevent metastases of patient triple negative tumors. Citation Format: Koichi Ito, Sun Hee Park, Anupma Nayak, Hanna Y. Irie. Novel dual regulation of epithelial-mesenchymal transition and anoikis resistance prevents metastases of triple negative breast cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B22.

Abstract P3-04-13: Protein tyrosine kinase 6 (PTK6) promotes survival of endocrine therapy-resistant ER+ breast cancer cells

Abstracts: 2016 San Antonio Breast Cancer Symposium; December 6-10, 2016; San Antonio, Texas Background/Rationale : The non-receptor tyrosine kinase PTK6/Brk is highly expressed in the ER+/Luminal breast cancer subtypes. PTK6 expression has prognostic significance for patients with ER+ disease; higher transcript levels are associated with poorer survival. The functions of PTK6 in the context of ER+ breast cancer and sensitivity to endocrine therapy have not been explored. We sought to determine the functional roles of PTK6 in ER+ breast cancer cells, including those that are relatively resistant to current endocrine therapies. Methods/Results. We modulated the expression of PTK6 using both gain- and loss-of-function approaches. Enhanced expression of PTK6 in Tamoxifen-sensitive ER+ breast cancer cells was sufficient to confer relative Tamoxifen resistance. Furthermore, downregulation of PTK6 in ER+ breast cancer cells, including those that have acquired resistance to Tamoxifen, induced apoptosis, as evidenced by an increase in AnnexinV+ cells and increased levels of cleaved PARP. PTK6 downregulation impaired growth of Tamoxifen-resistant variants of ER+ MCF7 and T47D cells (MCF7TamR and T47DTamR) in 3D Matrigel culture, and virtually abrogated primary tumor growth of MCF7TamR xenografts. Mechanistically, p38MAPK activation is critical for PTK6 downregulation-induced apoptosis of ER+ breast cancer cells, as p38 inhibition partially rescues cells from PTK6 shRNA-associated apoptosis. Conclusions: Our studies highlight the critical role that PTK6 plays in the survival of ER+ breast cancer cells, including those that are resistant to endocrine therapy. Enhanced PTK6 expression in ER+ breast cancer cells is sufficient to promote endocrine therapy resistance, which could contribute to the poorer prognosis associated with higher PTK6 expression in ER+ patient tumors. As small molecule PTK6 inhibitors are becoming available, our studies support further evaluation of PTK6 as a candidate therapeutic target for endocrine therapy resistant ER+ breast cancers. Citation Format: Irie HY, Park SH, Katsyv I, Zhang W, Nayak A. Protein tyrosine kinase 6 (PTK6) promotes survival of endocrine therapy-resistant ER+ breast cancer cells [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 P3-04-13.