Investigating molecular mechanisms of tumor eradication by targeting a major tumor vulnerability downstream of the EGFR/HER2/K-RAS signaling pathway: Seven in Absentia Homolog (SIAH)
Abstract
Introduction: Hyperactivation of the EGFR/K-RAS pathway is a major driver of multidrug-resistance, tumor progression, and metastatic dissemination in human malignancy that leads to poor clinical outcome and reduced overall survival. Designing effective therapies to contravene the intertwined, compensatory downstream effector pathways of EGFR/K-RAS signaling to ultimately achieve curative antitumor efficacy has proven difficult. Seven in absentia homologues (SIAH1 and SIAH2) are RING-domain E3 ubiquitin ligases that function as the most downstream signaling gatekeeper of the EGFR/RAS pathway. Our prior studies demonstrated that SIAH inhibition led to a dramatic tumor eradication phenotype of multiple stage IV human cancers cell lines in mouse xenograft models. We propose that SIAH is a major tumor vulnerability and actionable drug target for inhibiting persistent EGFR/K-RAS pathway activation that's responsible for driving tumor malignancy and metastasis. 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: State-of-the-art reverse phase protein arrays (RPPAs) in conjunction with Principal Component Analysis (PCA) were used to quantify fold-changes of 15-45 signaling proteins/phospho-proteins that were significantly up- or down-regulated in response to SIAH inhibition (p < 0.001). Independent RPPA assays 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 without DOX (group A) and with DOX induction (group B); Tet-ON-SIAH2PD cancer cells without DOX, [no SIAH2PD inhibitor expression] (group C) and with DOX induction [SIAH2PD inhibitor expression] (group D). The ratios of D/C/B/A, D/C, D/B, C/A, and B/A were calculated in a pairwise comparison after normalization to GAPDH as an internal control. To validate putative targets of interest, immunoblotting and fluorescence-activated cell sorting (FACS) analyses of biological triplicate cell lysates for each respective cell line were performed for group C and D at 3-, 5-, and 7-days post DOX (+) induction; target proteins' expression normalized to β-actin, α-Tubulin, or GAPDH respectively, and the pathway alterations induced by SIAH2 inhibition were standardized, quantified, and validated in SIAH-mutant cancer cells. Statistical analyses were performed by paired and unpaired student t-tests using the Prism software.
Results: Following the RPPA analyses in the five human cancer cell lines, we focused on these 7 putative target proteins: cleaved PARP, cleaved Caspase-3, cleaved Caspase-7, NFκB, phospho-Cofilin, PD-L1, and Collagens. Their altered protein expression was differentially detected in SIAH-proficient and SIAH-deficient cancer cell lines in a pairwise comparison. Performed Western blot analyses and FACS assays confirmed that cleaved PARP, cleaved Caspase-3, and cleaved Caspase-7 are markedly upregulated in SIAH-deficient cancer cells, suggesting a role in cell death and DNA damage pathway activation induced by SIAHloss of function as a novel mechanism of tumor suppression in human cancer.
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 target therapies to control and eradicate undruggable and relapsed human cancers in the future.