Tang, Amy

Abstract Hyperactive K-RAS signaling is a major menace that drives aggressive cancer cell dissemination, tumor progression and metastasis in human pancreatic cancer. Currently, there are no effective therapies to control human pancreatic 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 activation is an urgent goal and major challenge in targeted therapy development in pancreatic cancer. Instead of targeting an upstream signaling module such as EGFR/K-RAS/B-RAF/MEK/MAPK/ERK/AKT/mTOR, we targeted the most downstream signaling module in the K-RAS signaling 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 K-RAS signal transduction. Guided by the insights and principles learned from Drosophila RAS signaling pathway, we have conducted preclinical studies to dissect SIAH function in the context of K-RAS-mediated tumorigenesis and metastasis in human pancreatic cancer cells and tumor specimens. We found that inhibiting SIAH function is highly effective to abolish well-established and late-stage pancreatic tumor growth and metastasis in our pre-clinical studies. These findings demonstrate that SIAH is indeed an attractive, logical and potent anti-K-RAS therapeutic target for us to develop new and effective anticancer strategy against human pancreatic cancer. Through our work, SIAH has emerged as a new and effective drug target against oncogenic K-RAS hyperactivation in human pancreatic cancer. As one of the most evolutionarily conserved E3 ligases, SIAH is ideally and logically positioned to become a next-generation anti-K-RAS drug target in human cancer. Our preclinical studies have demonstrated that “SIAH-dependent proteolysis” is indeed an Achilles’ heel for human pancreatic cancer cells. Hence, anti-SIAH-based small molecule inhibitors may aid in expanding our arsenal of novel anticancer therapies in pancreatic cancer. By attacking the most downstream “gatekeeper” critical for the proper oncogenic ERBB/K-RAS signaling transmission, we may be in a position to halt the genesis, progression and metastasis of the most aggressive and the deadliest forms of human pancreatic cancer in the future. Citation Format: Vasilena Zheleva, Minglei Bian, Xiaofei Gao, Justin J. Odanga, Monicah M. Njogu, Zena M. Urban, Bruce E. Knudsen, Richard A. Hoefer, Roger R. Perry, Amy H. Tang. Developing a new and potent anti-K-RAS strategy by inhibiting SIAH E3 ligase, the most downstream “gatekeeper” in the oncogenic K-RAS signal pathway, to block well-established pancreatic tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B70.

Abstract The oncogenic EGFR/HER2/K-RAS “pathway” activation is pivotal in driving uncontrolled cell proliferation, aggressive tumor growth and systemic metastasis. Guided by studies in Drosophila, SIAH (seven-in-absentia homologue), the most downstream gatekeeper required for proper RAS signaling and tumor cell survival, was identified as a suitable target for anti-neoplastic therapy. We have shown that anti-SIAH-based therapy leads to the complete blockade of tumorigenesis in highly aggressive pancreatic, lung and breast cancer cells expressing a proteolysis deficient mutant protein, SIAHPD, at the onset of tumor initiation. In this study, we aim to determine if this anti-SIAH strategy is effective against well-established and late-stage pancreatic and triple negative breast (TNBC) tumors in a xenograft mouse model in vivo. We established stable cancer cell lines with doxycycline (DOX)-inducible SIAH2PD expression. One million MiaPaCa-SIAH2PD or MDA-MB-231-SIAH2PD cells were injected subcutaneously bilaterally in the dorsal scapular areas or bilaterally into the mammary fat pads, respectively, in 4-week-old female athymic nude mice. Tumor growth was monitored 3 times per week. Once tumors reached certain volume, SIAH2PD expression was induced via oral DOX treatment. Tumor growth in these mice was compared to control. Immunohistochemical (IHC) and immunofluorescence (IF) staining for endogenous SIAH and FLAG-tagged exogenous SIAH2PD were performed on formalin-fixed, paraffin-embedded sections of tumor tissue. Expression of SIAH2PD blocks oncogenic K-RAS signaling and K-RAS-mediated tumor growth in established pancreatic (MiaPaCa) and triple negative breast (MDA-MB-231) tumors in athymic nude mice. The immunohistochemical (IHC) and immunofluorescent (IF) staining reveals that in control tumors there is high expression of endogenous SIAH and none of SIAH2PD, while in tumors responding to treatment there is pronounced SIAH2PD and diminished endogenous SIAH expression. Oncogenic HER2/EGFR/K-RAS activation promote neoplastic transformation and tumorigenesis. In this study, we have identified a new vulnerability in the oncogenic K-RAS signaling pathway and we have demonstrated a novel and effective way to block K-RAS-driven tumor growth in well-established pancreatic adenocarcinoma and TNBC tumors in a xenograft model. In the follow-up studies, we hope to develop a small molecule inhibitor against SIAH, test their efficacy against patient-derived stage IV and II tumors in our “xenopatient” models. In the future, we aim to translate these exciting findings to the cancer clinics to benefit our cancer patients with metastatic diseases in VA. Citation Format: Minglei Bian, Vasilena Zheleva, Xiaofei Gao, Justin Odanga, Monica Njogu, Zena Urban, Bruce Knudsen, Richard A. Hoefer, Roger R. Perry, Amy H. Tang. Inhibition of late-stage tumor growth of human pancreatic and triple-negative breast cancer by blocking the most downstream “gatekeeper” signaling module, SIAH E3 ligase, in the oncogenic ERBB/ K-RAS signaling pathway. [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 4366. doi:10.1158/1538-7445.AM2015-4366

Abstract Oncogenic K-RAS activation is a major menace that drives aggressive tumor progression and metastasis in 30% of all human cancer. Currently, there are no effective therapies to treat stage III and IV metastatic human cancers with oncogenic K-RAS hyperactivation that often 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 pathway activation is an urgent goal and the major challenge in cancer therapy and anti-K-RAS-based drug development. Instead of targeting an upstream signaling module such as EGFR/HER2/K-RAS/B-RAF, we targeted the most downstream signaling module in the oncogenic K-RAS signaling pathway called the SIAH-dependent proteolytic machinery. SIAHs are the human homologs of Seven-In-Absentia (SINA), an evolutionarily conserved RING E3 ligase - the most downstream signaling module and a critical “gatekeeper” required for proper RAS signal transduction. Guided by the insights and fundamental principles learned from the Drosophila RAS signal transduction, we conducted preclinical studies to dissect SIAH function in promoting the oncogenic K-RAS-driven tumorigenesis and metastasis in human cancer. We found that (1) SIAH is a new biomarker reflective of oncogenic K-RAS activation in human cancer, and (2) SIAH loss-of-function is highly effective to block tumorigenesis and metastasis against the well-established, end-stage and metastatic pancreatic cancer and triple negative breast cancer (TNBC). These findings demonstrate that SIAH is an attractive and logical new therapeutic target for developing novel and effective anti-K-RAS and anticancer therapy against metastatic human cancer. Through our work, SIAH has emerged as a promising new drug target against oncogenic K-RAS hyperactivation in metastatic human cancer cells. Using anti-SIAH molecules to block oncogenic 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 that is the most downstream and the most conserved “signaling gatekeeper” in the oncogenic K-RAS signaling network, SIAH is uniquely and strategically positioned to become a great and logical anti-K-RAS drug target. Our preclinical studies have demonstrated that “SIAH-dependent proteolysis” is indeed an Achilles' heel in metastatic human cancer cells. Knowledge gained from our preclinical study has promising translational values. Anti-SIAH-based small molecule inhibitors are likely to aid in expanding our limited arsenal of novel anti-K-RAS-based anticancer therapies. By attacking the oncogenic K-RAS pathway using multi-pronged 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 position to control the late-stage, relapsed and metastatic human cancers by shutting down the hyperactivated K-RAS signaling transduction cascades in cancer cells in the future. Citation Format: Minglei Bian, Yang Liao, Vasilena Zheleva, Zena Urban, Monicah Njogu, Justin J. Odanga, Andrew J. Isbell, Roger R. Perry, Richard A. Hoefer, Thomas C. Smyrk, Gloria M. Petersen, Amy H. Tang. Attacking the most downstream “gatekeeper,” the SIAH-dependent proteolytic machinery, in the oncogenic ERBB/K-RAS signaling pathway to block tumorigenesis and control metastasis in human cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A26. doi: 10.1158/1557-3125.RASONC14-A26

Abstract Hyperactivation of the oncogenic K-RAS pathway leads to increased cell proliferation, tumor growth, and oxidative stress associated with increased ROS production, heightened mitochondrial bioenergetics, and altered cancer metabolisms. Downstream of the K-RAS pathway, Seven-In-Absentia human Homolog (SIAH) is required for proper ERBB/RAS signal transduction. Human peroxiredoxin (PRDX) is an antioxidant protein, whose precise role in modulating oxidative stress during oncogenesis remains to be elucidated. Increased levels of oxidative stress alter the bioenergetics and functional integrity of mitochondrial biology under cellular stress. This study aims to elucidate the biological interaction previously detected between PRDX and SIAH2 via biochemical assays. We plan to investigate the functional role of SIAH2-PRDX interaction in modulating the stress response and adaptive cell survival in K-RAS-driven tumors. Also, we would like to investigate the biological significance of SIAH2-PRDX interaction in response to increased cellular stress in K-RAS-driven human cancer cells treated with standard chemotherapies. Mitochondrial tracking (MITO-tracker®) was used to evaluate mitochondrial integrity of SIAH-proficient and SIAH-deficient human lung (A549) and cervical (HeLa) cancer cells over six days. Immunofluorescence staining and bright field imaging were performed using DOX-inducible human breast (MDA-MB231) and cervical (HeLa) cancer cells to compare changes in organelle integrity and mitochondrial morphology, altered expression of the oxidative stress biomarker and cell death biomarkers in response to induction of the SIAH- proficient and SIAH-deficient states in highly aggressive human cancer cells. By using human pancreatic cancer tissue microarrays (TMAs), we found that the antioxidant protein PRDX is significantly upregulated and co-expressed with SIAH in human pancreatic tumor biospecimens. It seems that PRDX is an indicative biomarker of cellular and oncogenic K-RAS-induced oxidative stress in human pancreatic cancer. By blocking SIAH2 function downstream of the K-RAS signaling pathway, oxidative stress levels were markedly increased, evidenced by mitochondrial aggregations and dysfunction in both human lung and cervical cancer cells. In response to anti-SIAH2 therapy, human TNBC, NSCLC and cervical cancer cells underwent dramatic morphological changes due to the significant loss of cell junction, focal adhesion and cell attachment. These data have shown that anti-SIAH2-based anti-K-RAS and anticancer therapy is highly effective in inducing massive cell death in several highly invasive and metastatic cancer lines. Further investigation into the molecular mechanisms of anti-SIAH2-induced cellular stress and cell death may contribute to the future drug development and validation of a new and effective anti-SIAH2-based targeted therapy against metastatic human cancer in the future. Citation Format: Zena M. Urban, Lauren van Reesema, Minglei Bian, Thomas C. Smyrk, Gloria Peterson, Amy H. Tang. Inhibition of SIAH E3 ligase function leads to increased cell death in metastatic human cancer cells. [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 4442. doi:10.1158/1538-7445.AM2014-4442

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 The pivotal roles of the K-RAS and TGFβ signaling pathways have been well characterized in a number of normal and pathological conditions including human cancer. Hyperactivation of the ERBB/KRAS pathway is known to drive neoplastic transformation, tumorigenesis and metastasis in a large percentage of human cancers while the growth inhibitory actions of TGFβ are critical in preventing cancer. A better understanding of the antagonistic interaction and cross-talk between the K-RAS and TGFβ signaling pathways that control tumorigenesis and metastasis may give rise to novel therapeutic interventions. Our previous work has highlight the importance of SIAH2 in K-RAS-dependent tumorigenesis and metastasis in human cancer. In this study, we showed that SMAD2/3/4 – the essential downstream mediators of TGFβ signaling, are new SIAH2 substrates. SIAH2 physically interacts with, ubiquitinates and degrades SMAD2/3/4 (but not SMAD1 – the downstream mediator of the BMP signaling pathway). The identification of the SIAH2-mediated SMAD2/3/4 modulation/degradation may provide an additional mechanism to explain the well documented, but less understood ERBB/K-RAS and TGFβ antagonism described in normal development, tumorigenesis and metastasis. We propose that the tumor promoting activity of RAS/SIAH2 may be mediated through the degradation of SMAD2/3/4 proteins and thus the relief of TGFβ-dependent growth inhibition in response to ERBB/K-RAS activation. The discovery of the SIAH2-SMAD2/3/4 antagonism downstream of K-RAS and TGFβ signaling pathways may provide a novel mode of antagonistic cross-talk between these two important signaling pathways to explain the growth inhibitory action of the TGFβ pathway and the growth-promoting activity of the K-RAS pathway in the genesis, progression and metastasis of human cancer. Since the substrate specificity of ubiquitin-mediated proteolysis is primarily determined by the E3 ligase, our results provide new insights into the role of altered proteolysis in mediating the context-dependent ERBB/RAS-TGFβ antagonism and cross-talk in 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 1980. doi:10.1158/1538-7445.AM2011-1980

Abstract The dismal prognosis of patients diagnosed with metastatic cancer points to our limited arsenal of effective anticancer and anti-metastasis therapies. Chemotherapy, radiation and targeted therapies are largely ineffective against metastatic cancer, resulting in the deaths of more than half million cancer patients each year. Oncogenic EGFR/HER2/K-RAS/B-RAF pathway activation is a major menace that drives aggressive cancer cell dissemination, tumor invasion and systemic metastasis. Therefore, finding novel approaches and new drug targets to inhibit hyperactive EGFR/HER2/K-RAS/B-RAF signaling is an urgent goal and major challenge in the struggle against metastatic diseases. Guided by the insights and principles learned from the evolutionarily conserved RAS signal transduction cascade in Drosophila, we targeted the most downstream signaling module in the EGFR/HER2/K-RAS/B-RAF signaling pathway, the SIAH-dependent proteolytic machinery. SIAHs are the human homologs of Seven-In-Absentia (SINA), a highly conserved RING E3 ligase, an essential downstream signaling module and a critical “gatekeeper” required for proper EGFR/HER2/K-RAS/B-RAF signal transduction. We found that inhibiting SIAH function is highly effective in halting cancer cell dissemination, altering cell adhesion and motility, inhibiting tumor invasion and cancer metastasis of human cancer cells. Importantly, our anti-SIAH-based anticancer strategy is effective in reducing tumor burdens in the late-stage and aggressive tumor growth and metastasis in the preclinical models. These exciting results and new findings demonstrate that SIAH is a highly attractive, mechanistically logical and great therapeutic target for developing novel anti-ERBB/K-RAS/B-RAF and anticancer therapies against invasive and metastatic cancer. Through our work, SIAH has emerged as a new, potent and promising drug target against metastatic diseases. As a highly evolutionarily conserved E3 ligase and the most downstream “gatekeeper” of EGFR/HER2/K-RAS/B-RAF signaling pathway, SIAH is ideally and logically positioned to become a next-generation anticancer and anti-metastasis drug target in the clinic. By simultaneously attacking the tumor-driving and metastasis-promoting ERBB/K-RAS signaling pathway using multi-pronged synergistic inhibitions at upstream (EGFR/HER2 membrane receptors), midstream (K-RAS/B-RAF/MEK/mTOR) and downstream (SIAH E3 ligase) signaling modules, we aim to make a difference and save lives from the deadliest forms of human metastatic cancers in the future. Citation Format: Amy H. Tang, Yang Liao, Minglei Bian, Vasilena Zheleva, Zena M. Urban, Monicah M. Njogu, Xiaofei Gao, Oscar A. Gonzalez, Justin J. Odanga, Bruce E. Knudsen, Roger R. Perry, Richard A. Hoefer, Thomas C. Smyrk, Gloria M. Petersen. Make a difference and save lives in human metastatic cancer by attacking the Achilles heel in the oncogenic ERBB/K-RAS signaling pathway. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A88.

Abstract Hyperactive ERBB/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 late-stage and relapsed human cancers that have oncogenic ERBB/K-RAS mutations that often confer drug resistance, aggressive tumor growth, systemic metastasis, and poor clinical outcome. Therefore, finding novel approaches and new drug targets to inhibit oncogenic ERBB/K-RAS activation 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, 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 EGFR/HER2/K-RAS/B-RAF signal transduction. Guided by the insights and fundamental principles learned from the 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 human cancer biospecimens. We found that (1) SIAH is a great prognostic and predictive biomarker for human cancer, and (2) inhibiting SIAH function is highly effective to block tumorigenesis and metastasis against the well-established, late-stage and metastatic pancreatic cancer and triple negative breast cancer (TNBC). These findings demonstrate that SIAH is an attractive and logical new therapeutic target for novel anti-ERBB/oncogenic K-RAS and anticancer therapy in human cancer. Through our work, SIAH has emerged as a new and effective drug target against oncogenic ERBB/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-ERBB/anti-K-RAS drug target. Our preclinical studies have demonstrated that “SIAH-dependent proteolysis” is indeed an Achilles' heel in human cancer. Knowledge gained from our study has great promise and 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 multi-pronged 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 position to control the late-stage, relapsed and metastatic human cancers. If successful, we hope to develop anti-SIAH-based therapy and translate our findings to the cancer clinics to treat cancer patients with metastatic diseases in the near future. Citation Format: Minglei Bian, Yang Liao, Vasilena Zheleva, Xiaofei Gao, Zena M. Urban, Monicah M. Njogu, Justin J. Odanga, Andrew J. Isbell, Rebecca L. Schmidt, Rie Takahashi, Roger R. Perry, Richard A. Hoefer, Thomas C. Smyrk, Gloria M. Petersen, Amy H. Tang. Attacking the most downstream “gatekeeper” signaling module in the oncogenic ERBB/K-RAS signaling pathway to block tumorigenesis and metastasis in human cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C62.

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 Breast cancer is the most common form of cancer diagnosed and the second leading cause of cancer death among women in the United States. Cancer metastases are the major cause of morbidity and mortality among breast cancer patients. The central importance of the HER2/RAS signaling pathway has been well established in promoting tumor growth, invasion and metastasis of human breast cancer. Even though oncogenic K-RAS mutations are rare in breast cancer, activation of the growth-promoting ERBB/RAS pathway has been consistently documented in high-grade breast tumors. As such, novel approaches to inhibit activated HER2/RAS signals constitute important measures to block tumor growth and metastasis in mammary tumors. In this study, instead of targeting an upstream signaling component such as HER2/EGFR/RAS, we targeted the most downstream signaling module identified in the ERBB/RAS pathway – the SIAH proteolytic machinery. SIAH is an evolutionarily conserved RING E3 ligase that is a critical “gatekeeper” required for RAS signal transduction in human cancer cells. SIAH is specifically expressed in tumor cells in 120 human breast cancer patients examined so far. The increased SIAH expression is correlated with increased grades and aggressiveness of human breast cancer. We asked whether inhibiting the gatekeeper function of SIAH would be effective in blocking mammary tumorigenesis and metastasis in human breast cancer. Our results indicated that SIAH2-deficiency successfully abolished tumor growth of three breast cancer cell lines (MDA-MB-231, MDA-MB-435 and MDA-MB-468) in soft agar assays as well as in athymic nude mice. Importantly, by inhibiting SIAH2 function in highly aggressive human breast cancer cells, we are able to completely block tumorigenesis and metastasis of two highly invasive and metastatic human breast cancer cell lines (MDA-MB-231 and MDA-MB-435). These results suggest that SIAH2 may be an attractive and new drug target for novel breast cancer therapy. Developing anti-SIAH molecules will aid in expanding our arsenal of effective anticancer therapies. By attacking the growth-promoting HER2/EGFR/RAS pathway at multiple signaling modules simultaneously – a multi-pronged synergistic inhibition at upstream (HER2/EGFR membrane receptors), midstream (RAS/RAF/MEK/mTOR) and downstream (SIAH E3 ligase) signaling modules in the HER2/EGFR/RAS pathway in parallel, we may be in a position to halt the genesis, progression and metastasis of human breast cancer. Thus, SIAH-dependent proteolysis may be an Achilles heel for breast cancer. SIAH inhibition may represent an innovative and logical way to inhibit HER2/EGFR/K-RAS activation, alleviate tumor burdens, halt cancer metastasis, and improve the efficacy for the future treatment of breast cancer. 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 1659. doi:10.1158/1538-7445.AM2011-1659