(F-228) FAK and AKT Signaling Regulate Mechanical Memory in Oral Squamous Cell Carcinoma

Introduction:: During tumor metastasis, cells must detach from a stiffened tumor and invade through softer stroma that surrounds them. For the cells to maintain their invasiveness throughout the metastasis, we hypothesized that cells need to acquire mechanical memory from tumor microenvironment and that the memory should persist. Our prior work suggested that Oral Squamous Cell Carcinoma (OSCC) can acquire memory,¹ and here we show that key transcriptomic and phosphoproteomic differences between cells with vs. without mechanical memory drive their functional differences and suggest memory durability.


Materials and Methods:: Cal27 was cultured in high glucose DMEM and supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were conditioned for 5 days in either soft (0.48kPa) or stiff (20kPa) (Learn) polyacrylamide gel coated with collagen. During the conditioning on stiff substrate, cells were treated with AKT inhibitor (Don’t Learn) to inhibit the biological pathway that we hypothesized to be involved in memory acquisition. After 5 days of conditioning, cells were moved to soft gel to be incubated for another 24-96 hours. During this replating phase, FAK inhibitor was added (Forget) to inhibit FAK pathway which is hypothesized to be involved in memory recovery. For immunostaining, E-cadherin and NMM2A were stained with primary and secondary antibodies to be fluorescently imaged.

Results, Conclusions, and Discussions:: To determine the pathways involved in memory acquisition, we performed a phospho-proteomic screen of 43 phosphorylation sites when Cal27 epithelial cells are “learning” to be mesenchymal in stiffer matrix or when they are challenged to “recall” their new mesenchymal identity in softer matrix. Analysis showed the greatest differential expression in the AKT and FAK pathways during “learning” and “recall” respectively (Fig. 1A). RNA sequencing between “Learn” (i.e., memory) vs. “Don’t Learn/Forget” (i.e., pharmacological inhibitors of AKT and FAK, respectively) mapped GO terms related to cell migration/motility and cell adhesion (Fig. 1B). Higher expression of non-muscle myosin 2 A (NMM2A) (Fig. 1C) and faster migration speed than those whose memory acquisition was inhibited validated these pathway screens. The trend in which cells conditioned on stiff substrate demonstrated higher cell migration speed than those on soft was maintained for 96 hours after replating (Fig. 1D), implying durability for downstream signals.


As OSCC learn from microenvironment stiffness, they acquire mechanical memory via AKT and then FAK to enhance contractility/motility as their niche change. Memory also appears durable.


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References (Optional): : 1. Matte et al, J Cell SCience (2019)