Triple negative breast cancer (TNBC) is a highly metastatic disease that currently lacks effective prevention and treatment strategies. The insulin-like growth factor 1 receptor (IGF1R) and focal adhesion kinase (FAK) signaling pathways function in numerous developmental processes, and alterations in both are linked with a number of common pathological diseases. Overexpression of IGF1R and FAK are closely associated with metastatic breast tumors. The present study investigated the interrelationship between IGF1R and FAK signaling in regulating the malignant properties of TNBC cells. Using small hairpin RNA (shRNA)-mediated IGF1R silencing methods, we showed that IGF1R is essential for sustaining mesenchymal morphologies of TNBC cells and modulates the expression of EMT-related markers. We further showed that IGF1R overexpression promotes migratory and invasive behaviors of TNBC cell lines. Most importantly, IGF1R-driven migration and invasion is predominantly mediated by FAK activation and can be suppressed using pharmacological inhibitors of FAK. Our findings in TNBC cells demonstrate a novel role of the IGF1R/FAK signaling pathway in regulating critical processes involved in the metastatic cascade. These results may improve the current understanding of the basic molecular mechanisms of TNBC metastasis and provide a strong rationale for co-targeting of IGF1R and FAK as therapy for mesenchymal TNBCs.

Phenotypic heterogeneity exists within collectively invading packs of tumor cells, suggesting that cellular subtypes cooperate to drive invasion and metastasis. Here, we take a chemical biology approach to probe cell:cell cooperation within the collective invasion pack. These data reveal metabolic heterogeneity within invasive chains, in which leader cells preferentially utilize mitochondrial respiration and trailing follower cells rely on elevated glucose uptake. We define a pyruvate dehydrogenase (PDH) dependency in leader cells that can be therapeutically exploited with the mitochondria-targeting compound alexidine dihydrochloride. In contrast, follower cells highly express glucose transporter 1 (GLUT1), which sustains an elevated level of glucose uptake required to maintain proliferation. Co-targeting of both leader and follower cells with PDH and GLUT1 inhibitors, respectively, inhibits cell growth and collective invasion. Taken together, our work reveals metabolic heterogeneity within the lung cancer collective invasion pack and provides rationale for co-targeting PDH and GLUT1 to inhibit collective invasion.

Tumor heterogeneity drives disease progression, treatment resistance, and patient relapse, yet remains largely underexplored in invasion and metastasis. Here, we investigated heterogeneity within collective cancer invasion by integrating DNA methylation and gene expression analysis in rare purified lung cancer leader and follower cells. Our results showed global DNA methylation rewiring in leader cells and revealed the filopodial motor MYO10 as a critical gene at the intersection of epigenetic heterogeneity and three-dimensional (3D) collective invasion. We further identified JAG1 signaling as a previously unknown upstream activator of MYO10 expression in leader cells. Using live-cell imaging, we found that MYO10 drives filopodial persistence necessary for micropatterning extracellular fibronectin into linear tracks at the edge of 3D collective invasion exclusively in leaders. Our data fit a model where epigenetic heterogeneity and JAG1 signaling jointly drive collective cancer invasion through MYO10 up-regulation in epigenetically permissive leader cells, which induces filopodia dynamics necessary for linearized fibronectin micropatterning.

Lung cancer is the leading cause of cancer-related deaths in the US with an overall 5-year survival of only 15-20%. Metastatic disease is a major contributor to poor survival and novel therapeutics that can selectively target invasive tumor cell populations are lacking. Metabolic reprogramming contributes to tumor progression with effective therapies targeting cancer metabolism available. However, it remains unclear whether targeting cancer metabolism will prove valuable in inhibiting invasive cancer populations. Recent studies have demonstrated anticancer activity by the mitochondria-targeting bioactive small molecule alexidine dihydrochloride. Our group has previously found that alexidine specifically inhibits the a highly invasive subpopulation of H1299 previous identified in our lab by decoupling and decreasing collective invasion. Here we report the structure activity relationship (SAR) between analogs of alexidine on the inhibition of collective invasion in NSCLC cell lines. For these studies, we utilized an in vitro 3D invasion model where NSCLC cell lines were allowed to form spheroids, and then were embedded into Matrigel and allowed to invade over 48h in the presence or absence of the analogs. Using this technique, we identified two novel alexidine analogs with improved efficacy at inhibiting the invasion of H1299 NSCLC cells. Additionally, we examined the phosphorylation of pyruvate dehydrogenase (PDH), the gatekeeper for the entrance of pyruvate into the TCA cycle, following treatment with the alexidine analogs. We observed a significant correlation between PDH phosphorylation and inhibition of invasion following treatment with the analogs. In conclusion, we have identified two novel alexidine analogs with improved inhibition of invasion in NSCLC cell lines in an in vitro model. Citation Format: Jamie Arnst, Rachel Commander, Adam Marcus. Structure activity relationship of alexidine analogs for the inhibition of collective invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1239.

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