Bian, Minglei

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely “undruggable”. Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future.

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 metastasized and oncogenic K-RAS mutations reman “undruggable” in the clinics. Therefore, counteracting K-RAS hyperactivation in attempt to reverse malignant transformation and inhibit latent tumor spread is an important goal in pancreatic cancer biology 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 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 and new drug target for anti-K-RAS and anticancer therapy. 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. Thus, we have identified a new vulnerability, SIAH E3 ligase, in the oncogenic K-RAS signaling pathway in pancreatic cancer. 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 pancreatic cancer. Our preclinical results 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 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. We aim to translate these exciting findings to the cancer clinics to benefit our pancreatic cancer patients with metastatic diseases in the future. Citation Format: Amy H. Tang, Minglei Bian, Vasilena Zheleva, Monicah M. Njogu, Justin J. Odanga, Roger R. Perry, Richard A. Hoefer, Bruce E. Knudsen.{Authors}. An effective new strategy to control and inhibit the “undruggable” oncogenic K-RAS hyperactivation in human pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B63.

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 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 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 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.