Targeted depletion of PIK3R2 induces regression of lung squamous cell carcinoma.

Oncogenic mutations in the PI3K/AKT pathway are present in nearly half of human tumors. Nonetheless, inhibitory compounds of the pathway often induce pathway rebound and tumor resistance. We find that lung squamous cell carcinoma (SQCC), which accounts for ~20% of lung cancer, exhibits increased expression of the PI3K subunit PIK3R2, which is at low expression levels in normal tissues. We tested a new approach to interfere with PI3K/AKT pathway activation in lung SQCC. We generated tumor xenografts of SQCC cell lines and examined the consequences of targeting PIK3R2 expression. In tumors with high PIK3R2 expression, and independently of PIK3CA, KRAS, or PTEN mutations, PIK3R2 depletion induced lung SQCC xenograft regression without triggering PI3K/AKT pathway rebound. These results validate the use PIK3R2 interfering tools for the treatment of lung squamous cell carcinoma.

E-cadherin downregulation sensitizes PTEN-mutant tumors to PI3Kß silencing.

Alterations in phosphatidylinositol 3-kinase (PI3K) and in PTEN (phosphatase and tensin homolog), the negative regulator of the PI3K pathway, are found in nearly half of human tumors. As PI3Kß, the main isoform activated in PTEN-mutant tumors, has kinase-dependent and -independent activities, we compared the effects of depleting vs. drug-inhibiting PI3Kß kinase activity in a collection of diverse tumor types and in a set of bladder carcinoma cell lines grown as xenografts in mice. PI3Kß depletion (by intratumor injection of PIK3CB siRNA) induced apoptosis and triggered regression of PTEN-mutant tumors more efficiently than PI3Kß inhibition. A small proportion of these tumors was resistant to PI3Kß downregulation; we analyzed what determined resistance in these cases. Using add-back experiments, we show that both PTEN mutation and low E-cadherin expression are necessary for PI3Kß dependence. In bladder carcinoma, loss of E-cadherin expression coincides with N-cadherin upregulation. We found that PI3Kß associated with N-cadherin and that PIK3CB depletion selectively disrupted N-cadherin cell adhesions in PTEN-mutant bladder carcinoma. These results support the use of PIK3CB interfering RNA as a therapeutic approach for high-risk bladder cancers that show E-cadherin loss and express mutant PTEN.

Phosphoinositide 3-kinase p85beta regulates invadopodium formation.

The acquisition of invasiveness is characteristic of tumor progression. Numerous genetic changes are associated with metastasis, but the mechanism by which a cell becomes invasive remains unclear. Expression of p85ß, a regulatory subunit of phosphoinositide-3-kinase, markedly increases in advanced carcinoma, but its mode of action is unknown. We postulated that p85ß might facilitate cell invasion. We show that p85ß localized at cell adhesions in complex with focal adhesion kinase and enhanced stability and maturation of cell adhesions. In addition, p85ß induced development at cell adhesions of an F-actin core that extended several microns into the cell z-axis resembling the skeleton of invadopodia. p85ß lead to F-actin polymerization at cell adhesions by recruiting active Cdc42/Rac at these structures. In accordance with p85ß function in invadopodium-like formation, p85ß levels increased in metastatic melanoma and p85ß depletion reduced invadopodium formation and invasion. These results show that p85ß enhances invasion by inducing cell adhesion development into invadopodia-like structures explaining the metastatic potential of tumors with increased p85ß levels.

Distinct and specific roles of AKT1 and AKT2 in androgen-sensitive and androgen-independent prostate cancer cells.

AKT isoforms are expressed in prostate cancer and their expression and localization have different associations with clinical characteristics. However, the distinct roles of the AKT isoforms in prostate cancer cells are largely unknown. In the present study, we demonstrate distinct roles for AKT1 and AKT2 in cell growth and migration. Ablation of AKT1 and AKT2 decreased the proliferation of the androgen-independent cell line PC-3, although by different mechanisms. AKT1 ablation induced loss of cell adhesion and subsequent apoptosis. AKT2 (but not AKT1) ablation promoted cell cycle arrest at G0/G1, associated with downregulation of cyclin D, CDK6 and CDK2, and upregulation and cytoplasmic-to-nuclear redistribution of p27. The increase of p27 protein levels was due to more gene transcription and an increase in protein stability. The increased stability of p27 was induced by delocalisation of Skp2 and a lower level of p27 phosphorylation at Thr187. AKT1 and AKT2 ablation inhibited and stimulated PC-3 cell migration, respectively. An AKT isoform-specific function could be associated with its subcellular localization. We found that AKT1 and AKT2 were mainly localised in the cytoplasm and nucleus, respectively. In androgen-sensitive cell line LNCaP, the ablation of AKT1 or AKT2 caused apoptosis but in androgen-independent LNCaP sublines, the effect of AKT1 ablation was lower; whereas no changes were observed after AKT2 ablation. Taken together, our data show that AKT1 and AKT2 have non-redundant roles in the regulation of PC-3 cell proliferation and migration. These could be explained by their subcellular localization and/or the specific regulation of downstream effectors. Furthermore, contribution of AKT isoforms to the progression of prostate cancer may change from an androgen-sensitive to a hormone-refractory stage. These findings may help design new targeted strategies for inhibiting AKT isoforms in prostate cancer.

EGF promotes neuroendocrine-like differentiation of prostate cancer cells in the presence of LY294002 through increased ErbB2 expression independent of the phosphatidylinositol 3-kinase-AKT pathway.

An increased neuroendocrine (NE) cell population in prostate cancer is associated with more aggressive disease and recurrence after androgen-deprivation therapy, although the mechanism responsible is unknown. In this study, we report that the treatment of LNCaP cells with epidermal growth factor (EGF) in the presence of LY294002, an inhibitor of the phosphoinositol 3'-kinase (PI3K)-AKT pathway, induced an increase of levels and activity of ErbB2. Under these conditions, we also observed cell survival and NE differentiation. When we treated with wortmannin, another PI3K inhibitor, or we knocked down PI3K or AKT isoforms in the presence of EGF, ErbB2 up-regulation was not observed, suggesting that the increase of ErbB2 induced by EGF plus LY294002 is not mediated by the PI3K-Akt pathway. Other targets of LY294002 were also discounted. We also show that ErbB2 up-regulation is directly involved in neuroendocine differentiation but not in cell survival as ErbB2 levels increased in parallel with NE differentiation marker levels, whereas ErbB2 knockdown reduced them; other NE differentiation inducers also increased the ErbB2 levels and the immunohistochemical analysis of prostate cancer samples showed colocalization of ErbB2 and chromogranin A. We found that, in LNCaP cells, EGF in combination with LY294002 increased ErbB2 levels by a PI3K/AKT-independent mechanism and that this increase was associated with the acquisition of a NE phenotype. These results suggest that is worth reconsidering ErbB2 as a drug target in prostate cancer and this should be kept in mind when designing new clinical schedules for the treatment of this disease.