PIK3C2B promotes epithelial to mesenchymal transition and EGFR inhibitors insensitivity in epidermal squamous cell carcinoma

While the class I of PI3Ks has been deeply studied due to its clear implication in cancer development, little is known about the class II of PI3Ks. However, recent accumulation of data is now revealing that PI3KC2β, one isoform of this class of PI3Ks, may also play a role in cancer. Specifically, recent studies have suggested an implication of PI3KC2β in metastasis formation through the promotion of epithelial to mesenchymal transition (EMT). Here, we report that the overexpression of PI3KC2β in the epidermal squamous cell carcinoma (ESCC) cells A431 promotes apparent EMT transformation. We further confirm this EMT by showing modification in several biochemical markers (E-cadherin, β-catenin, Snail, Twist1 and Vimentin). Furthermore, an intracellular co-localization of E-cadherin, β-catenin and EGFR was observed. This transformation decreased EGFR signaling and the sensitivity to inhibitors targeting this receptor. To confirm our results, we have used the colon adenocarcinoma cells HT29 and induced overexpression of PI3KC2β in these cells. We could recapitulate in this model some of our major findings regarding EMT in the PI3KC2β overexpressing A431 cells. Taken together, these data support a role of PI3KC2β in promoting EMT.

(12,13). However, the exact mechanism by which this kinase is participating in 93 this process is still unknown.

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In the epidermal squamous cell carcinoma (ESCC) cells A431, the 95 overexpression of PI3KC2β has been reported to enhance membrane ruffling, 96 migration speed of the cells, protection from anoikis and cell proliferation (14). 97 We decided to assess if some of these previously reported effects could be      Table 1.   Data is presented as average ± SD. All experiments were performed in triplicates.

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Statistical analyses were conducted using GraphPad Prism 7 (GraphPad  with E-Cadherin was also performed revealed partial co-localization of these 276 proteins in the cytoplasm (Fig 4A). 277 We performed here a dual-color immunofluorescence staining of EGFR 278 with E-cadherin ( Fig 4B). The overlap of the two signals in the cytoplasm showed to beta-Actin, since a strong increase of total-ERK was also found (Fig 5C).

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PI3KC2β has also been suggested to participate in chemo-resistance to 294 different drugs (6-8). We therefore tested if this PI3KC2β-driven relocalization of 295 EGFR translates into modified response to EGFR kinase inhibitors. Cell  (Fig 6A). Furthermore, HT29C2β cells also showed 315 increased mRNA levels for SNAI1 (2.5 folds) (Fig 6B). Finally, the comparison of 316 the morphology of the migration front between HT29 and HT29C2β cells showed 317 in HT29 an apparent tighter cell-to-cell contacts than in HT29C2β (Fig 6C). ). However, we were not able to determinate their specific cytoplasmic 347 localization as they do not co-localize with markers of Golgi apparatus or late 348 endosomes (Fig 3B-C). Despite this, the proximity to late endosomes markers 349 suggest that these molecules could be localized in another type of endosomes.

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A similar phenotype was observed in A431 after the treatment with 351 lysophosphatidic acid (LPA) (28). In this study E-cadherin and β-catenin were co- and being critical for cancer progression (28).

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In our study, the dual-colour immunofluorescence staining of PI3KC2β 359 with E-Cadherin revealed a partial co-localization of these proteins in a similar 360 discrete perinuclear region (Fig 4A). This supports the hypothesis that PI3KC2β 361 could be promoting the internalization of E-cadherin, β-catenin and EGFR.

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PI3KC2β is one of the major producers of PI3P in endosomes (29). Moreover, an stabilization of cell-cell adhesion and probably leading to EMT (Fig 7) (28).

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In addition to the observed effect in the cellular localization of EGFR, a 375 decrease in EGFR expression was also reported after PI3KC2β overexpression 376 (Fig 5B). This observation is in agreement with previous studies showing that in 377 A431 cells specific cellular context, EMT is also associated with a coordinated 378 loss of EGFR (30). Consistency with the decrease of EGFR, the phosphorylation 379 status of AKT and ERK, downstream targets of this receptor, was also decreased 380 ( Fig 5C). This reduction was less remarkable when compared to β-Actin levels, 381 since a strong increase of total-ERK was also found (Fig 5C), suggesting in 382 addition a possible compensatory mechanism.

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Together with the decrease in EGFR expression, a reduced sensitivity to 384 the Erlotinib and Gefitinib was also observed (Fig 5D). This is in agreement with 385 the transition to a mesenchymal-like phenotype that is known to decrease the 386 cellular dependence on EGFR signalling, as alternative growth pathways are 387 activated (31) (Fig 7). mRNA levels of Snail and decreased protein levels of E-Cadherin (Fig 6A-B). A 403 change in migration pattern decreasing cell-to-cell contact was also observed in 404 this cancer cell line (Fig 6C).     Snail. This constitutes the transcriptional machinery that will lead to EMT.