Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxisdifferential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments.

Targeted therapeutics that are initially effective in cancer patients nearly invariably engender resistance at some stage, an inherent challenge in the use of any molecular-targeted drug in cancer settings. In this study, we evaluated resistance mechanisms arising in metastatic melanoma to MAPK pathway kinase inhibitors as a strategy to identify candidate strategies to limit risks of resistance. To investigate longitudinal responses, we developed an intravital serial imaging approach that can directly visualize drug response in an inducible RAF-driven, autochthonous murine model of melanoma incorporating a fluorescent reporter allele (tdTomatoLSL). Using this system, we visualized formation and progression of tumors , starting from the single-cell level longitudinally over time. Reliable reporting of the status of primary murine tumors treated with the selective MEK1/2 inhibitor (MEKi) trametinib illustrated a time-course of initial drug response and persistence, followed by the development of drug resistance. We found that tumor cells adjacent to bundled collagen had a preferential persistence in response to MEKi. Unbiased transcriptional and kinome reprogramming analyses from selected treatment time points suggested increased c-Kit and PI3K/AKT pathway activation in resistant tumors, along with enhanced expression of epithelial genes and epithelial-mesenchymal transition downregulation signatures with development of MEKi resistance. Similar trends were observed following simultaneous treatment with BRAF and MEK inhibitors aligned to standard-of-care combination therapy, suggesting these reprogramming events were not specific to MEKi alone. Overall, our results illuminate the integration of tumor-stroma dynamics with tissue plasticity in melanoma progression and provide new insights into the basis for drug response, persistence, and resistance. A longitudinal study tracks the course of MEKi treatment in an autochthonous imageable murine model of melanoma from initial response to therapeutic resistance, offering new insights into the basis for drug response, persistence, and resistance. .

is overexpressed in multiple tumors, leading to the widely held view that this gene drives tumor progression, but this hypothesis has not been rigorously tested in melanoma. Here, we combined a conditional knockout of Coronin 1C with a genetically engineered mouse model of PTEN/ BRAF-driven melanoma. Loss of Coronin 1C in this model increases both primary tumor growth rates and distant metastases. Coronin 1C-null cells isolated from this model are more invasive in vitro and produce more metastatic lesions in orthotopic transplants than Coronin 1C-reexpressing cells due to the shedding of extracellular vesicles (EVs) containing MT1-MMP. Interestingly, these vesicles contain melanosome markers suggesting a melanoma-specific mechanism of EV release, regulated by Coronin 1C, that contributes to the high rates of metastasis in melanoma. Melanoma is the deadliest form of skin cancer with a high propensity for metastatic spread 1,2. The most common genetic drivers of melanoma are BRAF-activating mutations such as V600E, often found in conjunction with loss of tumor suppressors such as PTEN 3-5. If caught in its early stages, melanoma is treatable by surgical resection, but prognosis worsens significantly with the occurrence of in-transit, regional, or distant metastasis. Clinically, up to 54% of metastatic melanoma patients show tumor dissemination to the brain, 77% to the liver, and 85% to the lung 6. The high rate of metastasis in melanoma is poorly understood, but may be linked to lineage-specific factors that impact vesicular trafficking, secretion, degradation, and overall cell migration 7,8. The extracellular matrix (ECM) is an important substrate for tumor cell migration, but it also acts as a physical barrier whose degradation by matrix metalloproteinases (MMPs) is thought to be a critical step in tumor dissemination 9-11. MMPs are a family of both transmembrane (designated "membrane-type" or "MT") and secreted catalytic enzymes capable of cleaving ECM proteins and other substrates. Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a particularly important pro-invasive MMP across many cancer types 12 and whose expression closely correlates with invasion and metastasis 13-15. MT1-MMP is also unique in its ability to both directly cleave a wide range of ECM proteins including collagen types I, II, and III, laminin 1 and 5, and fibronectin, as well as activate pro-MMP2 16. The trafficking of MT1-MMP to and from the plasma membrane (PM) and other membrane structures is surprisingly complex, but critical for the protein's function during tumor invasion and metastasis. Phosphorylation of the cytoplasmic tail initiates internalization of MT1-MMP from the PM by both clathrin-mediated and caveolin-mediated routes 13. Internalized vesicles traffic through the endolysosomal pathway, resulting in redirection to other areas of the PM or to the lysosome for degradation 17. MT1-MMP recycling has been shown to involve various flotilins 18 and SNARE proteins 19-22 , as well as a variety of Rab proteins including Rab5, Rab7 1...

The G12/13 class of heterotrimeric G proteins, comprising the a-subunits Ga 12 and Ga 13 , regulates multiple aspects of cellular behavior, including proliferation and cytoskeletal rearrangements. Although guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) are well characterized as effectors of this G protein class, a variety of other downstream targets has been reported. To identify Ga 12 determinants that mediate specific protein interactions, we used a structural and evolutionary comparison between the G12/13, Gs, Gi, and Gq classes to identify "class-distinctive" residues in Ga 12 and Ga 13 . Mutation of these residues in Ga 12 to their deduced ancestral forms revealed a subset necessary for activation of serum response element (SRE)-mediated transcription, a G12/13-stimulated pathway implicated in cell proliferative signaling. Unexpectedly, this subset of Ga 12 mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of Ga 13 exhibited robust SRE activation, suggesting a Ga 12 -specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA-mediated lowering of Hsp90 levels resulted in greater downregulation of Ga 12 than Ga 13 signaling in SRE activation experiments. Furthermore, the Drosophila G12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and Ga 12 :Concertina chimeras revealed Ga 12 -specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify Ga 12 determinants of SRE activation, implicate Ga 12 :Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of Ga 12 and Ga 13 to allow bifurcated mechanisms of signaling to a common cell proliferative pathway.

<p>Achievement of reproducible spatial and temporal control of melanoma growth with <400 initiating cells</p>