Monicah M. Njogu

Abstract 5049: SIAH2 E3 ligase targets LIM-domain proteins for degradation to modulate focal adhesion and cell motility

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

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 3498: Increased cellular stress and apoptosis induced by anti-SIAH2 therapy in human cancer cells with oncogenic K-RAS addiction

Hyperactivated K-RAS oncoproteins are known to drive cell proliferation, tumor growth, and metastasis. Oncogene addiction is often associated with increased ER and mitochondrial stress due to excessive reactive oxygen and nitrogen species (ROS & NROS) production. RING finger E3 ubiquitin ligase, Seven in Absentia homolog 2 (Siah2) is a highly conserved, critical signaling module downstream of the EGFR/HER2/K-RAS pathway. SIAH2 function is required for proper K-RAS signal transduction and has been shown to target key regulators of oxidative stress for ubiquitination and proteasome degradation. However, the mechanisms by which SIAH2 regulates cellular stress and protects against programmed cell death in oncogenic K-RAS-addicted cancer cells are not well understood. Since SIAH2 is upregulated in proliferating tumor cells, we hypothesized that SIAH2-deficient cancer cells may be compromised in their ability to adapt to increased cellular and oxidative stress induced by oncogenic K-RAS pathway blockade. In this study, SIAH2 knockdown was carried out in cancer cells carrying oncogenic K-RAS mutations (A549, MDA-MB-231 and MiaPaCa cells). Triplicates of fresh cell lysates from SIAH2 proficient and SIAH2 deficient cells were then interrogated for altered expression of 19 signaling molecules with known functions in modulating stress response and apoptosis using the PathScan® Stress and Apoptosis Signaling Antibody Array kit. Differences in the protein expression were quantified and validated by Western blots, immunofluorescence (IF) and flow cytometry. We found that oxidative stress was markedly increased, evidenced by mitochondrial aggregations and dysfunction in a subset K-RAS-driven cancer cells, as we blocked SIAH2 function downstream of the oncogenic K-RAS signaling pathway. SIAH2 deficiency in breast cancer (MDA-MB-231) and non-small cell lung adenocarcinoma (A549) cells resulted in decreased ERK1/2 and AKT (S473) phosphorylation, increased ROS production, compromised mitochondria integrity and function, increased Caspase 3 and 7 activity, and PARP 1 cleavage that led to massive cell death observed in these cancer cells. In contrast, the prominent cell death was not observed in SIAH2 deficient pancreatic cancer (MiaPaCa) cells that expressed high levels of Survivin, which is an anti-apoptosis protein. The anti-SIAH2 therapy is highly effective in inducing oxidative stress, altered mitochondrial bioenergetics, and apoptosis in a subset of invasive and metastatic cancer lines expressing insufficient levels of Survivin. Our results suggest that, anti-SIAH2 strategy reveals novel oncogenic K-RAS-dependent cancer cell vulnerability in stress response that can be exploited in future for anti-SIAH2 therapy alone, or in combination with other anti-proliferative treatment to effectively combat KRAS-dependent cancers. Citation Format: Monicah M. Njogu, Caroline Lee, Jamie Eisner, Minglei Bian, Robert Vansciver, Amy H. Tang. Increased cellular stress and apoptosis induced by anti-SIAH2 therapy in human cancer cells with oncogenic K-RAS addiction. [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 3498.

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

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

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 Achilles9 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

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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

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

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.