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Cutaneous squamous cell carcinomas (SCCs) are commonly diagnosed skin cancers that may progress to invasiveness in the absence of early intervention. Using a murine model of SCC, we have previously demonstrated that activation of the Rho-associated kinase (ROCK) signaling pathway promotes rapid progression of pre-neoplastic lesions to invasive SCC. Herein we demonstrate that in human cutaneous SCC, ROCK signaling is increasingly up-regulated with tumor progression in both tumor cells and cells of the tumor microenvironment and is accompanied by key tumor-promoting changes in the extracellular matrix protein composition. The mechanotransduction pathway mediated by integrin signaling through FAK, GSK3β, and the transcription coactivator β-catenin is also progressively activated in human cutaneous SCC. Our observations indicate that ROCK activation is a tumor promoter in human cutaneous SCC and acts via mechanotransduction of signals to β-catenin. Our experiments raise the possibility that inhibition of ROCK signaling could be a useful therapeutic approach to halt cutaneous SCC progression by reducing the signal flux through this pathway to physiologic levels, thereby normalizing the extracellular matrix composition.

ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.

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While it is now well appreciated that the extracellular matrix (ECM) exerts biomechanical cues that direct critical cellular behavior, including cell proliferation, differentiation, migration, and survival, the molecular mechanisms underlying these cues remain mysterious. It has long been known that the ECM is also a source of biochemical cues that influence these processes, but the way these interact with ECM biomechanics also remains largely unknown. The systematic study of these relationships has been hampered by a paucity of models and the tools to interrogate them. Studies of complex models and tissue samples employing techniques such as atomic force microscopy (AFM) have informed much of our current understanding of how mechanical cues are transduced by the ECM and how cells respond to them. However, key observations made using such complex systems cannot be reliably assigned to the ECM or its components without a precise understanding of how these components respond to and exert mechanical force at the nanoscale – the scale at which individual cells respond. To address this knowledge gap, we used AFM to study the nanomechanical properties of a simple model, consisting only of type I collagen, the most abundant component of the ECM. Intriguingly, our data show bimodal distribution that is entirely attributable to type I collagen, greatly simplifying the interpretation of these studies. Furthermore, we examined the nanomechanical influence of tissue fixation by protein cross-linking, an approach commonly used in research and medical histopathology, revealing a significant and non-uniform distortion of the nanomechanical profile of fixed samples, which has the potential to introduce artifacts into the nanomechanical characterization of tissues. In contrast to the clear observation of mechanical differences induced by cross-linking, Fourier-transform infrared (FTIR) spectroscopy revealed only subtle alterations to the chemical signature of the collagen, highlighting the importance of nanomechanical approaches for the complete characterization of model systems and tissues.