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.

SIAH and EGFR, Two RAS Pathway Biomarkers, are Highly Prognostic in Locally Advanced and Metastatic Breast Cancer.

BACKGROUND: Metastatic breast cancer exhibits diverse and rapidly evolving intra- and inter-tumor heterogeneity. Patients with similar clinical presentations often display distinct tumor responses to standard of care (SOC) therapies. Genome landscape studies indicate that EGFR/HER2/RAS "pathway" activation is highly prevalent in malignant breast cancers. The identification of therapy-responsive and prognostic biomarkers is paramount important to stratify patients and guide therapies in clinical oncology and personalized medicine. METHODS: In this study, we analyzed matched pairs of tumor specimens collected from 182 patients who received neoadjuvant systemic therapies (NST). Statistical analyses were conducted to determine whether EGFR/HER2/RAS pathway biomarkers and clinicopathological predictors, alone and in combination, are prognostic in breast cancer. FINDINGS: SIAH and EGFR outperform ER, PR, HER2 and Ki67 as two logical, sensitive and prognostic biomarkers in metastatic breast cancer. We found that increased SIAH and EGFR expression correlated with advanced pathological stage and aggressive molecular subtypes. Both SIAH expression post-NST and NST-induced changes in EGFR expression in invasive mammary tumors are associated with tumor regression and increased survival, whereas ER, PR, and HER2 were not. These results suggest that SIAH and EGFR are two prognostic biomarkers in breast cancer with lymph node metastases. INTERPRETATION: The discovery of incorporating tumor heterogeneity-independent and growth-sensitive RAS pathway biomarkers, SIAH and EGFR, whose altered expression can be used to estimate therapeutic efficacy, detect emergence of resistant clones, forecast tumor regression, differentiate among partial responders, and predict patient survival in the neoadjuvant setting, has a clear clinical implication in personalizing breast cancer therapy. FUNDING: This work was supported by the Dorothy G. Hoefer Foundation for Breast Cancer Research (A.H. Tang); Center for Innovative Technology (CIT)-Commonwealth Research Commercialization Fund (CRCF) (MF14S-009-LS to A.H. Tang), and National Cancer Institute (CA140550 to A.H. Tang).

TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux.

A steady-state metaphase spindle maintains constant length, although the microtubules undergo intensive dynamics. Tubulin dimers are incorporated at plus ends of spindle microtubules while they are removed from the minus ends, resulting in poleward movement. Such microtubule flux is regulated by the microtubule rescue factors CLASPs at kinetochores and depolymerizing protein Kif2a at the poles, along with other regulators of microtubule dynamics. How microtubule polymerization and depolymerization are coordinated remains unclear. Here we show that TPX2, a microtubule-bundling protein and activator of Aurora A, plays an important role. TPX2 was phosphorylated by Aurora A during mitosis. Its phospho-null mutant caused short metaphase spindles coupled with low microtubule flux rate. Interestingly, phosphorylation of TPX2 regulated its interaction with CLASP1 but not Kif2a. The effect of its mutant in shortening the spindle could be rescued by codepletion of CLASP1 and Kif2a that abolished microtubule flux. Together we propose that Aurora A-dependent TPX2 phosphorylation controls mitotic spindle length through regulating microtubule flux.

Short exposure to paclitaxel induces multipolar spindle formation and aneuploidy through promotion of acentrosomal pole assembly.

Paclitaxel is a widely used microtubule drug and cancer medicine. Here we report that by short exposure to paclitaxel at a low dose, multipolar spindles were induced in mitotic cells without centrosome amplification. Both TPX2 depletion and Aurora-A overexpression antagonized the multipolarity. Live cell imaging showed that some paclitaxel-treated cells accomplished multipolar cell division and a portion of the daughter cells went on to the next round of mitosis. The surviving cells grew into clones with varied genome content. The results indicated that an aneuploidy population could be induced by short exposure to paclitaxel at a low dose, implicating potential side effects of paclitaxel.

Clathrin recruits phosphorylated TACC3 to spindle poles for bipolar spindle assembly and chromosome alignment.

Transforming acidic coiled-coil-containing protein 3 (TACC3) has been implicated in mitotic spindle assembly, although the mechanisms involved are largely unknown. Here we identify that clathrin heavy chain (CHC) binds specifically to phosphorylated TACC3 and recruits it to spindle poles for proper spindle assembly and chromosome alignment. Phosphorylation of Xenopus TACC3 at serine 620 (S620) and S626, but not S33, is required for its binding with CHC. Knockdown of CHC by RNA interference (RNAi) abolishes the targeting of TACC3 to spindle poles and results in abnormal spindle assembly and chromosome misalignment, similar to the defects caused by TACC3 knockdown. Furthermore, the binding of CHC with phosphorylated TACC3 is inhibited by importin ß and this inhibition is reversed by the presence of the GTP-binding nuclear protein Ran in the GTP-bound state. Together, these results indicate that the recruitment of phosphorylated TACC3 to spindle poles by CHC ensures proper spindle assembly and chromosome alignment, and is regulated by Ran.

A single amino acid change converts Aurora-A into Aurora-B-like kinase in terms of partner specificity and cellular function.

Aurora kinase-A and -B are key regulators of the cell cycle and tumorigenesis. It has remained a mystery why these 2 Aurora kinases, although highly similar in protein sequence and structure, are distinct in subcellular localization and function. Here, we report the striking finding that a single amino acid residue is responsible for these differences. We replaced the Gly-198 of Aurora-A with the equivalent residue Asn-142 of Aurora-B and found that in HeLa cells, Aurora-A(G198N) was recruited to the inner centromere in metaphase and the midzone in anaphase, reminiscent of the Aurora-B localization. Moreover, Aurora-A(G198N) compensated for the loss of Aurora-B in chromosome misalignment and cell premature exit from mitosis. Furthermore, Aurora-A(G198N) formed a complex with the Aurora-B partners, INCENP and Survivin, and its localization depended on this interaction. Aurora-A(G198N) phosphorylated the Aurora-B substrates INCENP and Survivin in vitro. Therefore, we propose that the presence of Gly or Asn at a single site assigns Aurora-A and -B to their respective partners and thus to their distinctive subcellular localizations and functions.

Roles of Aurora kinases in mitosis and tumorigenesis.

Aurora kinases, which have been implicated in several vital events in mitosis, represent a protein kinase family highly conserved during evolution. The activity of Aurora kinases is delicately regulated, mainly by phosphorylation and degradation. Deregulation of Aurora kinase activity can result in mitotic abnormality and genetic instability, leading to defects in centrosome function, spindle assembly, chromosome alignment, and cytokinesis. Both the expression level and the kinase activity of Aurora kinases are found to be up-regulated in many human cancers, indicating that these kinases might serve as useful targets for the development of anticancer drugs. This review focuses on recent progress on the roles of Aurora kinases in mitosis and tumorigenesis.