Investigating molecular mechanisms of tumor eradication by targeting a major tumor vulnerability downstream of the EGFR/K-RAS signaling pathway: Seven in Absentia Homolog (SIAH)

INTRODUCTION:
RAS GTPases represent the second most frequently mutated oncogenic driver in cancer, with more than 3 million new diagnoses per year. Harbored mutation hyperactivate the EGFR/K-RAS pathway subsequently dysregulating downstream signaling networks, driving cellular neoplastic transformation, tumorigenesis, treatment resistance, relapse, and metastatic dissemination, making K-RAS a major therapeutic target in the treatment of the most aggressive human cancers. Despite being studied for over 40 years, even the most recently FDA-approved small molecule inhibitors targeting K-RAS, have failed to reach curative anti-cancer efficacy: leaving oncogenic K-RAS as a largely "undruggable" target and unmet clinical need. Seven in absentia homologues (SIAHs) are RING-domain E3 ubiquitin ligases that function as the most downstream signaling gatekeeper of the EGFR/K-RAS pathway. Our preclinical studies demonstrated that SIAH inhibition led to a tumor eradication phenotype of multiple stage IV human cancer lines in xenograft models. We propose SIAH is a major tumor vulnerability and actionable drug target for inhibiting EGFR/K-RAS pathway activation. In this study, we aim to elucidate the molecular mechanisms underpinning the antitumor efficacy of our potent SIAH inhibitor as a promising new targeted therapy to achieve tumor eradication in vitro and in vivo.

METHODS:
Reverse phase protein arrays (RPPAs) in conjunction with Principal Component Analysis were used to identify significantly up- or down-regulated fold-changes of 294 signaling proteins/phospho-proteins in response to SIAH inhibition. RPPAs were performed in triplicate on doxycycline (DOX)-inducible MiaPaCa, MDA-MB-231, MDA-MB-468, HeLa, and A459 cell lines in which our SIAH inhibitor, SIAH2PD, expression was induced by a Tet-ON/OFF system. Four experimental conditions were used: Tet-ON control cells with/without DOX induction (group A/group B); Tet-ON-SIAH2PD cancer cells with/without DOX induction (group C-no inhibitor/group D-with inhibitor). The ratios of group were calculated in a pairwise comparison after normalization to GAPDH as an internal control. To validate putative targets of interest, immunoblotting, immunofluorescence, and fluorescence-activated cell sorting (FACS) were performed on cells for group C and D at 3-, 5-, and 7-days post-DOX-induction. Biological triplicate cell lysates were normalized to α-Tubulin, and adherent and single cell suspensions were used respectively for cell-based assays. Target proteins' expression was standardized, quantified, and validated in SIAH-mutant cancer cells. Statistical analyses were performed by paired and unpaired student ANOVA and t-tests using the Prism software.

RESULTS:
Following the RPPA analyses in the five human cancer cell lines, we focused on 7 putative target proteins: cleaved PARP, cleaved Caspase-3, cleaved Caspase-7, NFκB, Cofilin, PD-L1, and Collagens. Their altered protein expression was differentially detected in SIAH-proficient and SIAH-deficient cancer cell lines. Immunoblot, immunofluorescence, and FACS assays confirmed RPPA findings that cleaved PARP, cleaved Caspase-3, and cleaved Caspase-7, phospho-NFκB, and phospho-Cofilin are markedly upregulated in SIAH-deficient cancer cells, suggesting a role in cellular stress, DNA damage, and apoptosis pathway activation induced by SIAHloss of function.

CONCLUSION:
The RPPA-based cancer pathway mapping provides invaluable molecular insight into the antitumor efficacy of SIAH, revealing a major tumor vulnerability in human cancer network rewiring mechanisms when SIAH2 is blocked in late-stage, incurable cancer cells. The kinomic data support our innovative strategy to design anti-SIAH-based, anti-EGFR/K-RAS targeted therapies.