Walton, Zandra E.

Abstract Introduction: The RAS signaling pathway is an important growth-promoting pathway that controls cell proliferation and survival in multicellular organisms. During oncogenesis, activation of EGFR/HER2/K-RAS signaling pathway reduces focal adhesion, increases cell motility, promote tumorigenesis and accelerate tumor invasion and metastasis. SIAH2, which is the human homologue of a highly conserved family of RING-domain E3 ubiquitin ligase, is a key downstream signaling component which is essential for RAS signal transduction. We noticed that SIAH2 can regulate cell migration, tumorigenesis and metastasis by unknown mechanisms. Methods: Using HIV-based lentiviral system, we introduced an anti-SIAH molecule, the proteolysis-deficient mutant form of SIAH2 (SIAH2PD), into multiple types of human cancer cell lines. The changes of cell morphology, focal adhesion and adherens junction were examined by immunofluorescence (IF) staining. Images were captured on day 3 post infection experiments to show the changes in cell focal adhesion/junction. Using lentiviral system, we introduced exogenous LIM-domain proteins into human cancer cell lines which already expressed SIAH2PD, and then check the cell focal adhesion rescue by immunofluorescence staining. Results: We identified and validated three proteins that interact with SIAH2, contain LIM domains, and regulate focal adhesion and cell attachment. Through in vitro and in vivo experiments, we show that SIAH2 binds, ubiquitinates and degrades thyroid receptor-interacting protein 6 (TRIP6), Four-and-a-half LIM domain protein 2 (FHL2) and Leupaxin (LPXN), indicating that these evolutionarily conserved LIM-domain proteins are bona fide SIAH2 substrates and their double zinc finger motif might serve as a degron signal for SIAH2 E3 ligase. Our staining results showed that SIAH-deficiency would disrupt Trip6/FHL2/LPXN localization at the focal adhesion site, and inhibit cell motility. By introducing exogenous Trip6/FHL2/LPXN proteins into cancer cells, the focal adhesion deficiency and cell death caused by SIAHPD can be rescued, and the ability of cell migration and tumor initiation can be restored. Conclusions: We show that in cancer cells, SIAH2 antagonizes TRIP6, FHL2 and LPXN in modulating focal adhesions and cell motility. This interaction could be important in cell dissemination, invasion and metastasis. The suppression of cell motility and tumorigenesis in response to SIAH-deficiency could provide a new molecular mechanism to reveal the importance of RAS/SIAH signaling pathway in cancer cells. By circumventing this essential cell growth signaling pathway in cancer, we hope to develop novel anti-SIAH-based therapeutic drugs, which may help to inhibit cancer cell dissemination, invasion and metastasis. Citation Format: Minglei Bian, Yang Liao, Monicah Njogu, Rebecca Schmidt, Rie Takahashi, Zandra Walton, Amy H. Tang. SIAH2 E3 ligase targets LIM-domain proteins for degradation to modulate focal adhesion and cell motility. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5049.

Abstract Oncogenic EGFR/HER2/K-RAS activation is known to reduce focal adhesion and cell attachment, alter cell junction, and increase cell motility during normal tissue development as well as tumor invasion and cancer metastasis. Deciphering the mechanisms that promote cancer cell dissemination and metastasis in response to oncogenic ERBB/K-RAS “pathway” activation and finding novel targets for inhibiting invasion and metastasis remains an important goal in cancer biology. SIAH2, the human homologue of a highly conserved RING-domain E3 ubiquitin ligase SINA that is the most downstream signal modulator identified in Drosophila RAS signaling pathway, is critically required for proper mammalian ERBB/K-RAS signal transduction. SIAH2 insufficiency was shown to impede oncogenic K-RAS signal transduction, thereby obstructing K-RAS-driven tumorigenesis and metastasis in the preclinical models. However, the exact molecular mechanism of SIAH2 function downstream of the oncogenic ERBB/K-RAS activation remains to be characterized in human cancer cells. In this study, we use immunoprecipitation to isolate novel SIAH2-interacting proteins from multiple highly aggressive human cancer cells with oncogenic K-RAS hyperactivation. Three LIM-domain focal adhesion proteins (Trip6/FHL2/LPXN) were identified by Mass Spectrometry analysis, and their interactions with SIAH2 were verified biochemically by bi-directional co-immunoprecipitation (co-IP). Trip6/FHL2/LPXN has been reported to play important roles in cell focal adhesion formation and cell migration. We demonstrated that SIAH2 biochemically interacts with, ubiquitinates and thereby causes the degradation of these LIM domain proteins, demonstrating that TRIP6, FHL2 and LPXN are bona fide SIAH2 substrates in vivo. Our immunofluorescence (IF) staining results showed that SIAH2-deficiency disrupts Trip6/FHL2/LPXN localization at the focal adhesion site, results in defects in focal adhesion, cell junction, cell attachment and cell motility. Conversely, ectopic expression of TRIP6, FHL2 and LPXN can partially rescue SIAH2-deficient cancer cells from the pronounced defects in cell motility, cell death and tumor growth. The discovery that SIAH2 directly modulates focal adhesion may provide a novel molecular mechanism to explain the well-documented increases in cancer cell dissemination, tumor invasion and metastasis in response to ERBB/K-RAS pathway activation in human cancer cells. We may have uncovered a novel function of SIAH in modulating focal adhesion and cell junction in human cancer cells. By circumventing EGFR/HER2/K-RAS signaling pathway at its most downstream signaling module, we hope to validate and develop novel anti-SIAH-based anti-K-RAS anticancer therapies against oncogenic K-RAS-driven tumors in the future. Citation Format: Ming Bian, Yang Liao, Rebecca L. Schmidt, Monicah M. Njogu, Rie Takahashi, Zandra E. Walton, Amy H. Tang. Inhibiting SIAH2 E3 ligase function disrupts focal adhesion and cell junction, inhibits cell mobility and attachment, and blocks tumor invasion and metastasis in oncogenic K-Ras-driven tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1965. doi:10.1158/1538-7445.AM2014-1965

Abstract Hyperactivation of EGFR/HER2/K-RAS is often associated with reduced cell/focal adhesion, altered cell junctions, increased cell motility, and enhanced invasion/metastasis in a large percentage of human cancers. Finding novel measures that inhibit activated ERBB/K-RAS signals and, thus, reverse tumorigenesis and metastasis remains an important goal in cancer biology. Instead of targeting an upstream component in the EGFR/HER2/K-RAS pathway, we have targeted the most downstream signaling module, called the SIAH-dependent proteolytic machinery. SIAH/SINA are evolutionarily conserved RING E3 ligases that act as an essential “gatekeeper” signaling module in the RAS signal transduction pathway. We have demonstrated that proper SIAH function is required for K-RAS signal transduction. SIAH is a novel and tumor-specific biomarker for proliferating cells in human cancer. Furthermore, we have shown that SIAH2 inhibition can successfully block both tumorigenesis and metastasis in eleven of the most aggressive human cancer cell lines known in athymic nude mice. These observations highlight the importance of SIAH2 in K-RAS-dependent tumorigenesis and metastasis in human cancer. To elucidate the molecular mechanism of SIAH2 action in human cancer cells, a proteomic approach was taken to identify novel SIAH2-interacting proteins in three highly aggressive human cancer cell lines carrying K-RAS mutations (MiaPaCa, A549 and MDA-MB-435). From these studies, three focal adhesion LIM domain proteins (LPXN, TRIP6 and FHL2) were found to interact with SIAH2 endogenously in these human cancer cells. Here, we provide extensive biochemical evidence demonstrating that SIAH2 interacts with, ubiquitinates and degrades TRIP6/FHL2/LPXN in a dose-dependent fashion, both exogenously and endogenously. This study clearly demonstrates that LPXN, TRIP6 and FHL2 are bona fide SIAH2 substrates in vivo. Ectopic expression of LPXN/TRIP6/FHL2 successfully rescued the defects in cell motility and tumor growth observed in these SIAH2-deficient human cancer cells. These focal adhesion LIM domain proteins may, therefore, represent a novel class of SIAH2 substrates (logical RAS targets) that function downstream of SIAH2 and modulate focal adhesion, increased cell motility, invasion, tumorigenesis and metastasis in response to active ERBB/K-RAS signals. The identification of SIAH2 as an important regulator of focal adhesion and cell motility downstream of K-RAS pathway may help to explain the well-documented changes in cell adhesion, aggressive tumor growth, invasion and metastasis in response to K-RAS pathway activation in the most lethal forms of human cancers. 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 2335. doi:10.1158/1538-7445.AM2011-2335

Abstract Hyperactive K-RAS signaling is a major menace that drives aggressive tumor growth and metastasis in human cancer. Currently, there are no effective ways to treat human cancers that have oncogenic K-RAS mutations that confer drug resistance, aggressive tumor growth, systemic metastasis, and poor clinical outcome. Therefore, finding novel approaches and new drug targets to inhibit oncogenic K-RAS is an urgent goal and the major challenge in cancer therapy and treatment. Instead of targeting an upstream signaling module such as EGFR/HER2/KRAS/B-RAF/MEK/MAPK/AKT/mT0R, we targeted the most downstream signaling module in the EGFR/HER2/K-RAS pathway called the SIAH-dependent proteolytic machinery. SIAHs are the human homologs of seven-in-absentia (SINA), an evolutionarily conserved RING E3 ligase, an essential downstream signaling module, and a critical gatekeeper required for proper ERBB/RAS/RAF signal transduction. Guided by the insights and principles learned from Drosophila RAS signaling pathway, we conducted preclinical studies to dissect SIAH function in the context of ERBB/K-RAS-mediated tumorigenesis and metastasis in human cancer cells and tumor specimens. We found that: 1) SIAH is a novel biomarker for human cancer, i.e., SIAH specifically decorates proliferating tumor cells (not stromal or immune cells in the tumor microenvironment). SIAH expression is progressively and markedly upregulated as human cancer progresses to the advanced stages in human pancreatic, lung, breast, and prostate cancers; and 2) inhibiting SIAH function is highly effective to abolish tumorigenesis and metastasis in multiple of the most aggressive human cancer cell lines known in the literature that include pancreatic cancer cells (MiaPaCa and Panc-1), lung cancer cells (A549, H727, and UMC11), invasive breast cancer cells (MDA-MB-231, MDA-MB-468, and MDA-MB-435), colorectal cancer cells (SW620 and HCT116) and hormonal-refractory prostate cancer cells (PC-3 and DU-145) in our preclinical studies. These findings demonstrate that SIAH is an attractive and logical new therapeutic target for novel anti-K-RAS and anticancer therapy in human cancer. Through our work, SIAH has emerged as a new and effective drug target against oncogenic K-RAS activation in human cancer cells. Using anti-SIAH molecules to block K-RAS signaling in human cancer is an excellent example of science going from the bench (basic research in fruit flies) to the bedside (preclinical studies and ultimately clinical trials). As a highly evolutionarily conserved E3 ligase and the most downstream gatekeeper of EGFR/HER2/K-RAS/B-RAF signaling pathway, SIAH is uniquely and logically positioned to become a next-generation anti-K-RAS drug target. Our preclinical studies have demonstrated that SIAH-dependent proteolysis is an Achilles' heel for human cancer cells. Albeit at an early preclinical stage, knowledge gained from our study has great promise that has generated many excitements with immediate translational values. Anti-SIAH-based small molecule inhibitors may aid in expanding our arsenal of novel anticancer therapies. By attacking the oncogenic ERBB/K-RAS/B-RAF pathway using multipronged synergistic inhibitions at upstream (EGFR/HER2 membrane receptors), midstream (K-RAS/B-RAF/MEK/mTOR), and downstream (SIAH E3 ligase) signaling modules in parallel, we will be in a great position to halt the genesis, progression, and metastasis of the most aggressive and the deadliest forms of human cancers. If successful, we hope to translate our findings to the cancer clinics to treat many cancer patients with metastatic diseases in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B14.

Abstract The dismal prognosis of patients diagnosed with invasive and metastatic cancer points to our limited arsenal of effective anticancer therapies. The central importance of EGFR/HER2/RAS pathway activation has been well established in neoplastic transformation and tumorigenesis. An important goal in cancer biology is to identify novel and effective means to inhibit activated EGFR/HER2/K-RAS signals and reverse malignant transformation, tumor growth and cancer metastasis. SIAHs are the human homologs of Drosophila Seven-In-Absentia (SINA), an evolutionarily conserved RING E3 ligase that is an essential downstream signaling module and critical “gatekeeper” required for RAS signal transduction in human cancer cells. By targeting the most downstream signaling module identified in the RAS pathway - the SIAH2 proteolytic machinery, we are able to block tumorigenesis and metastasis in multiple of the most aggressive forms of human cancer cells known in athymic nude mice. Although we are fully cognizant of the systemic limitation in the xenograft models and incomplete mimicry of the full-spectrum metastatic processes observed in athymic nude mice, we are encouraged by the impressive anti-SIAH2-based tumor suppression in these preclinical studies. In this study, we aim to identify the molecular mechanism(s) of SIAH2 action in human cancer cells. To delineate SIAH2 function in cancer, through a proteomic approach, we identified three endogenous LIM domain proteins, TRIP6/FHL2/LPXN, as the bona fide SIAH2-interacting proteins isolated from the most aggressive human cancer cell lines (A549, MiaPaCa and MDA-MB-435 cells) in parallel. We provide extensive biochemical evidence to show that TRIP6/FHL2/LPXN are bona fide SIAH2 substrates. These new SIAH2 substrates can successfully rescue cell motility and viability defects observed in SIAH2-deficient cancer cells, suggesting that these focal adhesion proteins may represent one of many diverse signaling modules that function downstream of SIAH2 in mediating aspects of RAS-dependent signaling in normal development and cancer. Our results suggest In response to ERBB/RAS activation, SIAH2 E3 ligase modulates cell junction, adhesion and migration by targeting focal adhesion proteins for ubiquitin-mediated proteolysis to promote cell migration, tumorigenesis and metastasis in human cancer cells. The identification of SIAH2-mediated focal adhesion and cell junction modulation may have important implications in cancer biology to explain the well-documented modulations in focal adhesion, cell junction and motility in response to ERBB/RAS activation in human cancer cells. 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 4808.