Downstream Signaling Module Research Articles

Selexipag Improves Lipopolysaccharide-Induced ARDS on C57BL/6 Mice by Modulating the cAMP/PKA and cAMP/Epac1 Signaling Pathways.

Selexipag, a long-acting and selective prostacyclin (PGI2) IP receptor agonist, has in aged rats with stroke revealed effects of inhibiting inflammation, ameliorating damage to the blood-brain barrier, and alleviating oxidative stress. However, in the case of acute respiratory distress syndrome (ARDS) characterized by diffuse alveolar damage and lung capillary endothelial injury, its effects yet remain unknown. In this study, we investigated effects of the prophylaxis by Selexipag on a mouse model of ARDS established by the lipopolysaccharide (LPS) challenge and potential mechanism. Compared to the LPS-challenged mice, the LPS-challenged mice with the prophylaxis by 0.5 or 1 mg/kg of Selexipag exhibited significantly alleviated lung histological manifestations, reduced protein leakage, decreased levels of interleukin (IL)-1β, IL-6, and monocyte chemotactic protein-1 (MCP-1), diminished expressions of E-selectin and vascular cell adhesion molecule-1 (VCAM-1) mRNA, noticeably increased expressions of zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) protein, escalated lung cAMP levels, and raised levels of lung relative phosphorylated-protein kinase A catalytic subunit (p-PKA C) at Thr197 and exchange protein activated by cAMP 1 (Epac1) protein. These results suggest that, through suppressing inflammation and reducing vascular endothelial damage, Selexipag can effectively ameliorate the LPS-induced ARDS on mice. The lung cAMP and its downstream signaling modules, PKA and Epac1, possibly constitute the main regulative molecular mechanism. Selexipag appears to hold promise to become a new potential therapeutic option for ARDS.

Elevated ATP, cytokines and potential microglial inflammation distinguish exfoliation glaucoma from exfoliation syndrome

Glaucoma is the second leading cause of blindness. Exfoliation syndrome (XFN) is a risk factor for exfoliation glaucoma (XFG) which is a secondary open angle glaucoma. XFG is difficult to manage with a worse prognosis. Though 40% of the XFN progress to XFG, there are no predictive markers to identify the susceptible patients. Herein, we analyze clinical data, ATP levels in aqueous humor and cytokines in plasma to identify alteration that help distinguish XFN from XFG. Our results show characteristic clinical features of XFG compared to XFN and controls. Elevated levels of ATP in aqueous humor were observed in XFG compared to XFN and cataract controls while elevated levels of plasma cytokines were observed in XFG compared to XFN, cataract controls and healthy controls. Microglia are immune cells in the retina implicated in glaucoma. TNFα plays a predominant role in microglial inflammation and is implicated in neurodegeneration. Using in vitro N9 microglial cell culture model, we demonstrate that TNFα modulated expression of cytokines and chemotaxis is dependent on P2 receptors like P2X7, P2Y12 and P2Y6. In addition, ATP also induce expression of TNFα which might act as a feed forward loop. The TNFα induced inflammation is dependent on downstream signaling modules like PI3K, JNK and ROS. Taken together, our results show that elevated ATP in aqueous humor, plasma cytokines and inflammation potentially involving microglia distinguish XFG from XFN. Purinergic receptors might be potential therapeutic targets in XFG.

Abstract PS17-58: Pparg1 induces an EGF-EphA2 receptor tyrosine kinase module to promote ErbB2- mammary adenocarcinoma in mice

Abstract ErbB2 is overexpressed in approximately 25% of human breast cancers, associated with clinically aggressive disease. No soluble ligand has been identified and the receptor is regulated by heterodimerization with other ErbB family receptors, including EGFR, and other receptor tyrosine kinases including EphA2. EGFR is activated by seven different growth factors including EGF and Amphiregulin. Downstream signaling modules required for ErbB2 induced tumorigenesis in genetically engineered mouse models (GEMM) include the phosphatidylinositol 3-kinase/Akt (PKB) pathway, the Ras/Raf/MEK/ERK1/2 pathway and the phospholipase C (PLCγ) pathways. ErbB2-mediated tumorigenesis involves activation of receptor tyrosine kinases, induction of cyclin D1/CDK activity, and functional restraint by tumor suppressors. The receptor tyrosine kinase EPH receptor A2 (EphA2), a member of the Eph RTK family, is overexpressed in aggressive breast cancer and EphA2 forms a complex with ErbB2 thereby enhancing ErbB2-induced tumor onset and progression. The host immune system participates in the therapeutic response of HER2+ breast cancer. The tumor microenvironment (TME) is regulated by chemokines and their G protein coupled receptors binds several ligands, including Cxcl5 which binds Cxcr2, to augment the pro-tumor immune response, tumor growth and metastasis. Identifying genetic programs that participate in ErbB2-induced tumors may provide the rational basis for co-extinction therapeutic approaches. Peroxisome proliferator-activated receptor γ (PPARγ), which is expressed in a variety of malignancies, governs biological functions through transcriptional programs. Herein, genetic deletion of endogenous Pparγ1 restrained mammary tumor progression, lipogenesis, and induced local mammary tumor F4/80+ tumor-associated macrophage infiltration, without affecting other tissue hematopoietic stem cell pools. Pparγ1 induced peroxisomal target genes in the mammary tumors as evidenced by increased expression of PEX-11, together with PPARGC1 and ESRR induced regulator, muscle 1, Perm1 (PGC-1 and ERR-induced Regulator in Muscle 1). Peroxisomes induced by Pparγ1, activated Type1 interferons (IFNs) and IFN-stimulated gene expression, including Cxcl5. Endogenous Pparγ1 induced expression of both an EphA2-Amphiregulin and an inflammatory INFγ -Cxcl5 signaling module. Pparγ1 bound directly to growth promoting and proinflammatory target genes in the context of chromatin. We conclude Pparγ1 promotes ErbB2-induced tumor growth and inflammation and represents a relevant target for therapeutic coextinction. Citation Format: Richard G Pestell, Xuanmao Jiao, Andrew V Kossenkov, Adam Ertel, Wei Tong, Zhao Zhang, Peter A McCue. Pparg1 induces an EGF-EphA2 receptor tyrosine kinase module to promote ErbB2- mammary adenocarcinoma in mice [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-58.

DNA-based artificial molecular signaling system that mimics basic elements of reception and response

In order to maintain tissue homeostasis, cells communicate with the outside environment by receiving molecular signals, transmitting them, and responding accordingly with signaling pathways. Thus, one key challenge in engineering molecular signaling systems involves the design and construction of different modules into a rationally integrated system that mimics the cascade of molecular events. Herein, we rationally design a DNA-based artificial molecular signaling system that uses the confined microenvironment of a giant vesicle, derived from a living cell. This system consists of two main components. First, we build an adenosine triphosphate (ATP)-driven DNA nanogatekeeper. Second, we encapsulate a signaling network in the biomimetic vesicle, consisting of distinct modules, able to sequentially initiate a series of downstream reactions playing the roles of reception, transduction and response. Operationally, in the presence of ATP, nanogatekeeper switches from the closed to open state. The open state then triggers the sequential activation of confined downstream signaling modules.

Abstract 2145: Detect early tumor relapse in high-risk breast cancer

Abstract Chemotherapy resistant breast cancer is a major health challenge, resulting in high relapse rates and poor survival. Neoadjuvant chemotherapy (NACT) is a standard treatment for women with high-risk TNBC, HER2+, and locally advanced ER+ breast cancer. A completed course of NACT results in two possible outcomes: pathologic complete response (pCR) or residual disease. While pCR is a reliable clinical prognostic biomarker associated with excellent outcomes and prolonged survival, especially for TNBC and HER2+ cancers, patients with residual disease have a higher risk of recurrence. Patients with residual disease can be further stratified clinically using the Residual Cancer Burden (RCB) classification; however, patients with similar RCB classes may experience dramatically different clinical outcomes. Thus, additional precision biomarkers to stratify patients with residual disease after NACT are needed to identify patients at highest risk of recurrence, and to develop new therapeutic strategies to eradicate multidrug-resistant tumors. Supported by strong evidence in developmental, evolutionary and cancer biology, we found that K-RAS-SIAH pathway activation is a major tumor driver, and SIAH represents a key tumor vulnerability in breast cancer. Normal K-RAS/SIAH signaling pathway activation is indispensable for proper cell-cell communication, cell proliferation and tissue homeostasis in multicellular organisms. However, abnormal K-RAS/SIAH pathway activation is highly prevalent in high-risk and locally advanced breast cancer, and may confer chemo-resistance to these high-risk mammary tumors. Based on its evolutionary conservation and significance as the most downstream signaling module indispensable for K-RAS signal transduction, SIAHON/OFF expression is a reliable readout of K-RAS/EGFR/HER2 pathway activation/inactivation. We showed that SIAHON/OFF expression is a binary code in residual mammary tumors that can be used to stratify patients, augment RCB classification, forecast tumor relapse, and predict patient survival after 1st line NACT in a pilot retrospective study. Multidrug-resistant high-grade breast cancer is a genetically diverse, highly heterogeneous disease that challenges our ability to individualize and optimize precision therapy. Persistent K-RAS-SIAH-EGFR pathway activation endows TNBC with therapy resistance, and increases the risk of metastasis and early relapse. As such, we developed a K-RAS/SIAH-centered biomarker discovery program and a new anti-SIAH research initiative with the intended goal of designing novel and potentially life-saving anti-K-RAS targeted strategies to control and eradicate multidrug-resistant and intractable mammary tumors. Citation Format: Amy H. Tang, Lauren L. Siewertsz van Reesema, Vasilena P. Zheleva, Janet S. Winston, Rick J. Jansen, Emanuel F. Petricoin, Matthew P. Goetz, Matthew P. Goetz, Harry D. Bear, Richard A. Hoefer. Detect early tumor relapse in high-risk breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2145.

Abstract 83: The critical role of seven-in-absentia (SINA) family E3 ligases in normal development and oncogenic K-RAS-driven human cancer

Abstract There will be an estimated 1.8 million new cancer diagnoses in the US this year alone, with >80% of these cases driven by oncogenic K-RAS/SIAH pathway activation, even in the absence of oncogenic RAS mutations. Over three decades of intense research has focused on conquering RAS pathway activation in cancer, yet oncogenic K-RAS has remained essentially undruggable. Seven-IN-Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signaling pathway thus far. Underscoring SINA’s importance is its high degree of evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling “gatekeeper” in the RAS pathway, we have shown that SIAH is required for oncogenic K-RAS-driven tumorigenesis and metastasis in human pancreatic, lung, and breast cancers. Since SIAHs appear to be the ideal drug target to inhibit “undruggable” K-RAS activation, it is important to precisely characterize the activity, regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the Drosophila photoreceptor development system, we conducted a genetic modifier screen and identified 28 new sina mutant alleles that exhibit a range of mutant phenotypes. For example, sina complete loss of function (null alleles) is cell lethal, and several newly identified point mutant alleles exhibit stronger mutant phenotypes than those of previously published truncated alleles. Sequencing analysis of these sinamutant revealed critical roles of several immutable amino acid residues indispensable for SINA function. To demonstrate the functional conservation of SINA/SIAH proteins, we generated a collection of transgenic fly lines that carry either wild-type (WT) or dominant negative (DN) DmSINA and human SIAH1/2. Tissue-specific SINA/SIAH expression using GAL4 drivers revealed the biological consequences of gain or loss of function of SINA/SIAH1/2 in transmitting RAS signaling in Drosophila development. Immunofluorescent (IF) staining of developing imaginal discs and electron micrographs of adult tissues show that ectopic expression of SINAWT/DN/SIAHWT/DN in neurons resulted dramatic changes in neuronal cell fate in the developing eye and sensory neuronal cells on the notum. Furthermore, IF staining of larval salivary glands revealed a critical role for SINA/SIAH in regulating cell shape, focal adhesions, and cellular junctions. Results from these transgenic models suggest that Drosophila SINA and human SIAH1/2 are highly evolutionarily conserved and functionally interchangeable. Ultimately, we aim to translate these preclinical findings to demonstrate the high potency and anticancer efficacy of an anti-SIAH-based anti-K-RAS strategy against multidrug-resistant and incurable human cancer. Citation Format: Robert E. Van Sciver, Yajun Cao, Atique U. Ahmed, Amy H. Tang. The critical role of seven-in-absentia (SINA) family E3 ligases in normal development and oncogenic K-RAS-driven human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 83.

Dual inhibition of ERK1/2 and AKT pathways is required to suppress the growth and survival of endometriotic cells and lesions

Endometriosis is an estrogen-dependent and progesterone-resistant gynecological inflammatory disease of reproductive-age women. Current hormonal therapies targeting estrogen can be prescribed only for a short time. It indicates a need for non-hormonal therapy. ERK1/2 and AKT pathways control several intracellular signaling molecules that control growth and survival of cells. Objectives of the present study are to determine the dual inhibitory effects of ERK1/2 and AKT pathways: (i) on proliferation, survival, and apoptosis of human endometrioitc epithelial cells and stromal cells in vitro; (ii) on growth and survival of endometrioitc lesions in vivo in xenograft mouse model of endometriosis of human origin; and (iii) establish the associated ERK1/2 and AKT downstream intracellular signaling modules in the pathogenesis of endometriosis. Our results indicated that combined inhibition of ERK1/2 and AKT pathways highly decreased the growth and survival of human endometriotic epithelial cells and stromal cells in vitro and suppressed the growth of endometriotic lesions in vivo compared to inhibition of either ERK1/2 or AKT pathway individually. This cause-effect is associated with dysregulated intracellular signaling modules associated with cell cycle, cell survival, and cell apoptosis pathways. Collectively, our results indicate that dual inhibition of ERK1/2 and AKT pathways could emerge as potential non-hormonal therapy for the treatment of endometriosis.

A New Strategy to Control and Eradicate "Undruggable" Oncogenic K-RAS-Driven Pancreatic Cancer: Molecular Insights and Core Principles Learned from Developmental and Evolutionary Biology.

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.

Seven‐In‐Absentia (SINA) Family E3 Ligases in Development and Growth

The RAS signal transduction cascade is a pivotal signaling pathway that controls numerous fundamental cellular processes, such as cell proliferation, differentiation, motility, and apoptosis. Oncogenic K‐RAS pathway activation is detected in approximately 30% of all human cancers. Seven‐IN‐Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signaling pathway. Underscoring the importance of SINA is its high evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling hub and the downstream signaling “gatekeeper” in the RAS pathway, SIAHs are uniquely positioned to inhibit “undruggable” oncogenic K‐RAS activation that is prevalent in high‐grade metastatic cancer. Thus, it is important to delineate the enzymatic activity, molecular regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the elegant and powerful Drosophila photoreceptor cell development system, we conducted a genetic modifier screen and identified 28 new sina mutant alleles that exhibit a range of mutant phenotypes. For example, sina complete loss of function (null alleles) is cell lethal, suggesting that SINA is an essential gene in development. All other sina mutant alleles exhibit stronger phenotypes than the previously published sina2 and sina3 alleles, providing us with an opportunity to study sinaloss‐of‐function mutant phenotypes. Sequencing analysis of these newly identified mutant alleles reveals the critical roles of several immutatable amino acid residues essential for SINA function. To demonstrate the functional conservation of SINA/SIAH proteins, we have generated a collection of transgenic fly lines that carry either wild‐type (WT) or dominant negative (DN) DmSINA and human SIAH. The altered DmSINA/hSIAH expression under the control of sev‐, GMR‐, and dpp‐ and salivary gland‐GAL4 drivers revealed the biological consequences of gain of function and/or loss of function of DmSINA/hSIAH1/2 in transmitting RAS signaling in central nervous system (CNS) and peripheral nervous system (PNS) development. Immunofluorescent staining of developing imaginal discs and electron micrographs of adult tissues show that ectopic expression of SINAWT/DN/SIAHWT/DN in neurons resulted in dramatic changes in neuronal cell fate in the developing eye and notum. Furthermore, immunofluorescent staining of larval salivary glands revealed a critical role for SINA family E3 ligases in regulation of cell shape, focal adhesions, and cellular junctions. The results from these transgenic animals suggested that Drosophila SINA and human SIAH1/SIAH2 are evolutionarily conserved and functionally interchangeable. Through this developmental neurobiology and cellular biology‐based study, we aim to dissect the molecular action of SINA/SIAH1/2 in RAS signaling. Furthermore, we hope to translate these findings to demonstrate the anticancer efficacy of the next‐generation anti‐SIAH‐based anti‐K‐RAS strategy against high‐grade metastatic human cancer in the future.Support or Funding InformationExtramural funding support provided by NIH NIGMS R01 GM069922Z‐05S1 and NCI R01 CA140550.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 4824: The “gatekeeper” function of Drosophila Seven-IN-Absentia (SINA) E3 ligase and its human homologs, SIAH1 and SIAH2, is highly conserved for proper RAS signal transduction in Drosophila development

Abstract Seven-IN-Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signal transduction cascade. Underscoring the importance of SINA is its high evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling “gatekeeper” in the RAS pathway, we have shown that SIAH is required for oncogenic K-RAS-driven tumorigenesis and metastasis in human pancreatic, lung and breast cancer. Since SIAHs appear to be the ideal drug target to inhibit “undruggable” K-RAS activation, it is important to precisely characterize the activity, regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the elegant and well-established Drosophila development system, we are able to study RAS activation and SINA function under normal physiological conditions. In the developing Drosophila eye, photoreceptor cells are recruited sequentially and acquire their distinctive cell fates through a series of local inductive events. The 800x cell arrays allowed us to dissect the role of SINA/SIAH downstream of RAS activation in photoreceptor cell development. To delineate SINA function, we performed an F1 modifier screen using ethyl methanesulfonate (EMS) and X-ray radiation, isolating 28 novel sina mutant alleles. These mutant alleles exhibit much stronger mutant phenotypes than those of the previously published sina2 and sina3 alleles, suggesting that the sina2 and sina3 alleles are hypomorphic alleles. Sequencing analysis of these sinamutant alleles reveals the functional roles of mutated residues and protein domains. To define SINA/SIAH functional conservation, we have generated a complete panel of transgenic fly models that express either wild-type (WT) or dominant negative (DN) SINA/SIAH. The corresponding UAS-sina/siahGOF/LOF phenotypes have been characterized using sev-, GMR-, dpp- and salivary gland-GAL4 drivers to elucidate the developmental outcomes of altered SINA/SIAH expression upon RAS activation. Ectopic expression of sinaWT/DN/siahWT/DN in neurons resulted in dramatic changes in neuronal cell fate in the developing eye and notum, causing PNS neurodegenerative phenotypes. Our results show that the biological functions of fly SINA and human SIAH1/SIAH2 are evolutionarily conserved and functionally interchangeable. Mechanistic insights and regulatory principles learned from Drosophila can be directly applied to cancer biology to develop and validate next-generation anti-SIAH-based anti-K-RAS and anticancer therapy in the future. Citation Format: Robert E. Van Sciver, Yajun Cao, Atique U. Ahmed, Amy H. Tang. The “gatekeeper” function of Drosophila Seven-IN-Absentia (SINA) E3 ligase and its human homologs, SIAH1 and SIAH2, is highly conserved for proper RAS signal transduction in Drosophila development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4824. doi:10.1158/1538-7445.AM2017-4824

Abstract B63: An effective new strategy to control and inhibit the “undruggable” oncogenic K-RAS hyperactivation in human pancreatic cancer

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.

Neratinib induces ErbB2 ubiquitylation and endocytic degradation via HSP90 dissociation in breast cancer cells

Receptor tyrosine kinase ErbB2/HER2 is frequently observed to be overexpressed in human cancers, leading to over activation of downstream signaling modules. HER2 positive is a major type of breast cancer for which ErbB2 targeting is already proving to be an effective therapeutic strategy. Apart from antibodies against ErbB2, the small molecule tyrosine kinase inhibitor lapatinib has had successful clinical outcomes, and other inhibitors such as neratinib are currently undergoing clinical investigations. In this study we report the effects of lapatinib and neratinib on the mRNA and protein levels of the ErbB2 receptor. We provide evidence that neratinib-induced down regulation of ErbB2 occurs through ubiquitin-mediated endocytic sorting and lysosomal degradation. At the mechanistic level, neratinib treatment leads to HSP90 release from ErbB2 and its subsequent ubiquitylation and endocytic degradation. Our findings provide novel insights into the mechanism of ErbB2 inhibition by neratinib.

Abstract 2008: Proteomic analysis of SIAH2 E3 ligase complex in oncogenic K-Ras-driven cell transformation in human cancer

Abstract Affinity purification-mass spectrometry (AP-MS) is a highly effective method of identifying and quantifying the individual constituents of molecular machines that are dynamically regulated by diverse cellular stimuli during tumor initiation, progression and metastasis. Seven In absentia homolog 2 (SIAH2) is an evolutionarily conserved E3 ubiquitin ligase and a key component of a molecular machine called “SIAH2-dependent proteolytic machinery” that serves as the most downstream signaling module critical for proper oncogenic ERBB/RAS signal transduction. As a newly identified promising anti-K-RAS and anticancer drug target, it is important to understand the dynamic regulation of SIAH function, substrate recognition and complex formation during tumor initiation, progression and metastasis. Our aim was to delineate the molecular regulation, substrate selection and target recognition of the SIAH proteolytic machinery in K-Ras-driven cellular transformation and oncogenesis; and to explore the SIAH-dependent K-RAS oncogene addiction and its future therapeutic application. In this study, affinity purification was conducted in triplicates to isolate the SIAH2 protein complexes from two panels of human epithelial cells: For the lung epithelial cells: we used the normal bronchial epithelial cells (BEAS-2B), RAS-transformed BEAS-2B (BZR) and non-small cell lung cancer (NSCLC) A549 cells with oncogenic K-RAS activation. For the mammary epithelial cells: we used normal mammary epithelial cells (HMEC and MCF-10A), benign breast tumor cells (MCF-7), MDA-MB-231 that carries both oncogenic K-RAS and B-RAF activation and MDA-MB-468. The extracted proteins were trypsin-digested and peptides subjected to high throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. SIAH2 was observed to propagate downstream RAS signals by affecting proteins involved in cellular junctions, focal adhesion, metabolism, mitochondrial function and kinase activities. Using Western blots, immunofluorescence staining and co-immunoprecipitation assays, we have verified and confirmed that focal adhesion proteins, TRIP6 (Thyroid Receptor Interacting Protein 6) and FHL2 (Four and Half Lim domain protein) are SIAH2 substrates in human cancer cell lines lung cancer (A549) and triple negative breast cancer (MDA-MB-231) and breast cancer (MDA-MB-468). We found that TRIP6 primarily localizes to the focal adhesions, cell junctions and the nucleus of the cells. Overexpression of SIAH2 induces the degradation of exogenous and endogenous TRIP6 while Inhibition of SIAH2 enzymatic function results in a stabilization of TRIP6 and mis-localization of TRIP6 in human cancer cells. In conclusion, we report that SIAH2 regulates focal adhesion, cell junction and cellular attachment by down regulating TRIP6 expression levels and may induce a more motile and aggressive phenotype in human cancer cells. Citation Format: Monicah M. Njogu, Ming Lei Bian, Jamie Eisner, Amy H. Tang. Proteomic analysis of SIAH2 E3 ligase complex in oncogenic K-Ras-driven cell transformation in human cancer. [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 2008. doi:10.1158/1538-7445.AM2015-2008

Abstract B70: 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 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 A26: 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 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 1965: 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 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 4439: Proteomic analysis of SIAH2 E3 ligase complex in oncogenic K-Ras-driven cell transformation and oncogenesis

Abstract Affinity purification-mass spectrometry (AP-MS) is a highly effective method of identifying and quantifying the individual constituents of molecular machines that are dynamically regulated by diverse cellular stimuli during tumor initiation, progression and metastasis. Seven In absentia homolog 2 (SIAH2) is an evolutionarily conserved E3 ubiquitin ligase and a key component of a molecular machine called “SIAH2-dependent proteolytic machinery” that serves as the most downstream signaling module critical for proper oncogenic ERBB/RAS signal transduction. Our aim was to use a proteomic approach to identify differentially expressed SIAH2-interacting proteins isolated from normal epithelial cells, benign tumor and malignant cancer cells, and delineate their roles in K-Ras-driven cellular transformation and oncogenesis. In this study, affinity purification was conducted in triplicates to isolate the SIAH2 protein complexes from normal bronchial epithelial cells (BEAS-2B), RAS-transformed BEAS-2B (BZR) and oncogenic K-RAS activated NSCLC (A549) cells. The extracted proteins were trypsin-digested and peptides subjected to high throughput liquid chromatography tandem mass spectrometry (LC-MS/MS). The data was processed and used in searches against the latest versions of the SwissProt and NCBI protein databases. Peptide and protein identifications were filtered at a False Discovery Rate (FDR) of 1% using the decoy database strategy. Dynamic changes, post-translational modifications and altered expression of individual protein components in the SIAH2-complexes were identified and quantified in a pairwise comparison. Out of the proteins identified as putative SIAH2 interacting partners, twelve top hit SIAH2-interacting proteins were chosen for validation and further analysis. Using Western blots, immunofluorescence staining and co-immunoprecipitation assays, we have verified and confirmed that TRIP6 (Thyroid Receptor Interacting Protein 6) is a newly identified SIAH2 substrate in six human cancer cell lines (pancreatic cancer (MiaPaCa), lung cancer (A549), cervical cancer (HeLa), triple negative breast cancer (MDA-MB-231), breast cancer (MDA-MB-468), melanoma (SK-MEL-28). We found that TRIP6 primarily localizes to the focal adhesions, cell junctions and the nucleus of the cells. Overexpression of SIAH2 induces the degradation of exogenous and endogenous TRIP6 while Inhibition of SIAH2 enzymatic function results in a stabilization of TRIP6 and mis-localization of TRIP6 in human cancer cells. In conclusion, distinct changes of SIAH2 complexes were detected in normal epithelial, benign tumor and metastatic cancer cells. Our proteomic ID experiments are still ongoing. We have found that SIAH2 regulates focal adhesion, cell junction and cellular attachment by down regulating TRIP6 expression levels and may induce a more motile and aggressive phenotype in human cancer cells. Citation Format: Monicah M. Njogu, Ming Lei Bian, Amy H. Tang. Proteomic analysis of SIAH2 E3 ligase complex in oncogenic K-Ras-driven cell transformation and oncogenesis. [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 4439. doi:10.1158/1538-7445.AM2014-4439

Inhibition of Established Pancreatic and Triple Negative Breast Tumor Growth by Blocking the Most Downstream Signaling Module, SIAH, in the Oncogenic ERBB/K-RAS Signaling Pathway

Inhibition of Established Pancreatic and Triple Negative Breast Tumor Growth by Blocking the Most Downstream Signaling Module, SIAH, in the Oncogenic ERBB/K-RAS Signaling Pathway

Abstract C62: Attacking the most downstream “gatekeeper” signaling module in the oncogenic ERBB/K-RAS signaling pathway to block tumorigenesis and metastasis in human cancer

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 A88: Make a difference and save lives in human metastatic cancer by attacking the Achilles heel in the oncogenic ERBB/K-RAS signaling pathway

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.