Presentations

Abstract

Introduction:

RAS proteins are a family of evolutionarily conserved small GTPases that rest in the heart of a central tumor- driving pathway, the EGFR/HER2/RAS/RAF pathway, where they function as molecular switches, critically regulating cellular differentiation, proliferation, and survival. Its hyperactivation is a potent driver of neoplastic transformation and tumorigenesis found in ~40% of all human cancers, and is associated with highly aggressive, multidrug-resistant, recurrent, and metastatic cancers: making oncogenic RAS a major therapeutic target in the treatment of human cancers across multiple pathologies. However, despite 40 years of continued research, the adaptability, dynamic kinetics, and plasticity of downstream oncogenic signaling networks make K-RAS a largely "undruggable" target that has yet to achieve clinical efficacy. Seven in absentia homologues (SIAH1 and SIAH2) are evolutionarily conserved RING-domain E3 ubiquitin ligases that are expressed in proliferating cells; thus far, they have been identified as the most downstream signaling gatekeeper of the EGFR/HER2/RAS/RAF pathway. Our prior studies demonstrated that effective SIAH inhibition abolishes tumor growth in aggressive cancer cell lines, such as MiaPaCa, MDA-MB-231, MDA-MB-468, A459, and HeLa. We propose that SIAH is a major tumor vulnerability and actionable drug target for inhibiting oncogenic EGFR/HER2/RAS/RAF compensatory signaling network activation; we aim to elucidate the molecular mechanisms underpinning the anti-cancer "curative" phenotype of SIAH inhibitors.

Methods:

300 proteins/phospho-proteins were quantitatively measured by Reverse Phase Protein Array (RPPA) platforms to identify compensatory signaling pathway activation/inactivation and systemic cancer network rewiring in response to anti- SIAH blockade. Independent RPPAs were performed in triplicate on doxycycline (DOX)-inducible Tet-ON MiaPaCa, MDA- MB-231, MDA-MB-468, HeLa, and A459 cell lines were generated from a single cell, in which DOX-induced SIAH inhibitor, SIAH PD, expression was confirmed. Each cell line was then subjected to one of four experimental conditions: Tet- ON control cells without DOX induction (group A), Tet-ON control cells with DOX induction (group B), Tet-ON-SIAH2PD cancer cells without DOX induction (no SIAH PD inhibitor) (group C), Tet-ON-SIAH PD cancer cells with DOX induction (SIAH PD inhibitor) (group D). RPPAs, in conjunction with Principal Component Analysis (PCA), were conducted to quantify significant fold-changes of proteins/phosphoproteins whose expression was altered in response to SIAH inhibition (p ≤ 0.001). The ratios of D/C/B/A, D/C, D/B, C/A, and B/A were calculated after normalization to GAPDH expression as an internal control. Prism was used to compare altered protein expression in a pairwise comparison. Targets that were identified as significantly up- or down-regulated in response to SIAH blockade were then assessed by immunoblotting for validation. Cell lysates of biological quadruplicates for each respective cell line were collected for group C and group D at different time points, 3-, 5-, and 7-days post DOX induction; protein concentration of each lysate was determined by bicinchoninic acid assays, and the proteins of interest were then normalized to β-actin, α-Tubulin, or GAPDH (based on molecular weight), and DOX-induced changes in protein expression were standardized, quantified, and plotted.

Results:

Through RPPAs, we identified 6 targets (NFκB, Caspase-7, PARP, Cofilin, PD-L1, and Collagens) whose protein expression showed significant changes in response to SIAH blockade. These proteins play a critical role in regulating cell growth, apoptosis, NFκB signaling, stress response, DNA damage and repair mutants, immune dysfunction, and loss of cell adhesion in SIAH-deficient cancer cells. Immunoblot analyses yielded supporting evidence of our initial RPPA findings, providing independent validation of changes in the aforementioned targets' response to SIAH inhibition.

Conclusion:

The identification and validation of differential protein expression induced by SIAH blockade provides valuable molecular insight into the dynamic regulation underpinning SIAH inhibitors' unmatched anti-tumor efficacy against stage IV human tumors. Our emerging data provide clear evidence supporting SIAH as a major tumor vulnerability and an actionable target that can be used to control and eradicate undruggable and incurable human cancers in the future.