Tumor Biomechanics Alters Metastatic Dissemination of Triple Negative Breast Cancer via Rewiring Fatty Acid Metabolism.

In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin ß1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.

Memory of stochastic single-cell apoptotic signaling promotes chemoresistance in neuroblastoma.

Gene expression noise is known to promote stochastic drug resistance through the elevated expression of individual genes in rare cancer cells. However, we now demonstrate that chemoresistant neuroblastoma cells emerge at a much higher frequency when the influence of noise is integrated across multiple components of an apoptotic signaling network. Using a JNK activity biosensor with longitudinal high-content and in vivo intravital imaging, we identify a population of stochastic, JNK-impaired, chemoresistant cells that exist because of noise within this signaling network. Furthermore, we reveal that the memory of this initially random state is retained following chemotherapy treatment across a series of in vitro, in vivo, and patient models. Using matched PDX models established at diagnosis and relapse from individual patients, we show that HDAC inhibitor priming cannot erase the memory of this resistant state within relapsed neuroblastomas but improves response in the first-line setting by restoring drug-induced JNK activity within the chemoresistant population of treatment-naïve tumors.

In Vitro 3D Models of Tunable Stiffness.

Three-dimensional models of spheroid formation have been routinely used in the cancer field to test the colony forming capacity of malignant cells in an in vitro setting. Use of such a model provides a robust surrogate for in vivo testing, enabling large-scale interrogation into the effect of certain treatment conditions. This adapted protocol describes a high throughput and readily accessible composite alginate hydrogel system for spheroid formation, within a biomechanically tunable three-dimensional environment. This model therefore allows users to examine the effect of certain treatment conditions while cells are embedded within a hydrogel of defined stiffness. This is particularly important in the context of cancer where cells experience a wide range of mechanical properties within their microenvironment, driven by widespread changes in the extracellular matrix composition and architecture.This protocol describes a high-throughput method which results in homogeneous interpenetrating polymer networks of collagen and alginate. We show that this network readily supports single-cell spheroid formation in numerous malignant cell lines (breast cancer, lung cancer, and melanoma) and that these can be robustly analyzed for colony formation measures such as spheroid size, spheroid number, and overall cell viability; therefore, allowing users to undertake high-throughput, in vitro screening against a controlled biomechanical background.

A cryptic tubulin-binding domain links MEKK1 to curved tubulin protomers.

The MEKK1 protein is a pivotal kinase activator of responses to cellular stress. Activation of MEKK1 can trigger various responses, including mitogen-activated protein (MAP) kinases, NF-κB signaling, or cell migration. Notably, MEKK1 activity is triggered by microtubule-targeting chemotherapies, among other stressors. Here we show that MEKK1 contains a previously unidentified tumor overexpressed gene (TOG) domain. The MEKK1 TOG domain binds to tubulin heterodimers-a canonical function of TOG domains-but is unusual in that it appears alone rather than as part of a multi-TOG array, and has structural features distinct from previously characterized TOG domains. MEKK1 TOG demonstrates a clear preference for binding curved tubulin heterodimers, which exist in soluble tubulin and at sites of microtubule polymerization and depolymerization. Mutations disrupting tubulin binding decrease microtubule density at the leading edge of polarized cells, suggesting that tubulin binding may play a role in MEKK1 activity at the cellular periphery. We also show that MEKK1 mutations at the tubulin-binding interface of the TOG domain recur in patient-derived tumor sequences, suggesting selective enrichment of tumor cells with disrupted MEKK1-microtubule association. Together, these findings provide a direct link between the MEKK1 protein and tubulin, which is likely to be relevant to cancer cell migration and response to microtubule-modulating therapies.

Structure-based mechanism of preferential complex formation by apoptosis signal-regulating kinases.

Apoptosis signal-regulating kinases (ASK1, ASK2, and ASK3) are activators of the p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. ASK1-3 form oligomeric complexes known as ASK signalosomes that initiate signaling cascades in response to diverse stress stimuli. Here, we demonstrated that oligomerization of ASK proteins is driven by previously uncharacterized sterile-alpha motif (SAM) domains that reside at the carboxy-terminus of each ASK protein. SAM domains from ASK1-3 exhibited distinct behaviors, with the SAM domain of ASK1 forming unstable oligomers, that of ASK2 remaining predominantly monomeric, and that of ASK3 forming a stable oligomer even at a low concentration. In contrast to their behavior in isolation, the ASK1 and ASK2 SAM domains preferentially formed a stable heterocomplex. The crystal structure of the ASK3 SAM domain, small-angle x-ray scattering, and mutagenesis suggested that ASK3 oligomers and ASK1-ASK2 complexes formed discrete, quasi-helical rings through interactions between the mid-loop of one molecule and the end helix of another molecule. Preferential ASK1-ASK2 binding was consistent with mass spectrometry showing that full-length ASK1 formed hetero-oligomeric complexes incorporating large amounts of ASK2. Accordingly, disrupting the association between SAM domains impaired ASK activity in the context of electrophilic stress induced by 4-hydroxy-2-nonenal (HNE). These findings provide a structural template for how ASK proteins assemble foci that drive inflammatory signaling and reinforce the notion that strategies to target ASK proteins should consider the concerted actions of multiple ASK family members.

Targeting promiscuous heterodimerization overcomes innate resistance to ERBB2 dimerization inhibitors in breast cancer.

BACKGROUND: The oncogenic receptor tyrosine kinase (RTK) ERBB2 is known to dimerize with other EGFR family members, particularly ERBB3, through which it potently activates PI3K signalling. Antibody-mediated inhibition of this ERBB2/ERBB3/PI3K axis has been a cornerstone of treatment for ERBB2-amplified breast cancer patients for two decades. However, the lack of response and the rapid onset of relapse in many patients now question the assumption that the ERBB2/ERBB3 heterodimer is the sole relevant effector target of these therapies. METHODS: Through a systematic protein-protein interaction screen, we have identified and validated alternative RTKs that interact with ERBB2. Using quantitative readouts of signalling pathway activation and cell proliferation, we have examined their influence upon the mechanism of trastuzumab- and pertuzumab-mediated inhibition of cell growth in ERBB2-amplified breast cancer cell lines and a patient-derived xenograft model. RESULTS: We now demonstrate that inactivation of ERBB3/PI3K by these therapeutic antibodies is insufficient to inhibit the growth of ERBB2-amplified breast cancer cells. Instead, we show extensive promiscuity between ERBB2 and an array of RTKs from outside of the EGFR family. Paradoxically, pertuzumab also acts as an artificial ligand to promote ERBB2 activation and ERK signalling, through allosteric activation by a subset of these non-canonical RTKs. However, this unexpected activation mechanism also increases the sensitivity of the receptor network to the ERBB2 kinase inhibitor lapatinib, which in combination with pertuzumab, displays a synergistic effect in single-agent resistant cell lines and PDX models. CONCLUSIONS: The interaction of ERBB2 with a number of non-canonical RTKs activates a compensatory signalling response following treatment with pertuzumab, although a counter-intuitive combination of ERBB2 antibody therapy and a kinase inhibitor can overcome this innate therapeutic resistance.