Upregulation of Receptor Tyrosine Kinase Activity and Stemness as Resistance Mechanisms to Akt Inhibitors in Breast Cancer.

The PI3K/Akt pathway is frequently deregulated in human cancers, and multiple Akt inhibitors are currently under clinical evaluation. Based on the experience from other molecular targeted therapies, however, it is likely that acquired resistance will be developed in patients treated with Akt inhibitors. We established breast cancer models of acquired resistance by prolonged treatment of cells with allosteric or ATP-competitive Akt inhibitors. Phospho-Receptor tyrosine kinase (Phospho-RTK) arrays revealed hyper-phosphorylation of multiple RTKS, including EGFR, Her2, HFGR, EhpB3 and ROR1, in Akt-inhibitor-resistant cells. Importantly, resistance can be overcome by treatment with an EGFR inhibitor. We further showed that cancer stem cells (CSCs) are enriched in breast tumor cells that have developed resistance to Akt inhibitors. Several candidates of CSC regulators, such as ID4, are identified by RNA sequencing. Cosmic analysis indicated that sensitivity of tumor cells to Akt inhibitors can be predicted by ID4 and stem cell/epithelial-mesenchymal transition pathway targets. These findings indicate the potential of targeting the EGFR pathway and CSC program to circumvent Akt inhibitor resistance in breast cancer.

Upregulation of AKT3 Confers Resistance to the AKT Inhibitor MK2206 in Breast Cancer.

Acquired resistance to molecular targeted therapy represents a major challenge for the effective treatment of cancer. Hyperactivation of the PI3K/AKT pathway is frequently observed in virtually all human malignancies, and numerous PI3K and AKT inhibitors are currently under clinical evaluation. However, mechanisms of acquired resistance to AKT inhibitors have yet to be described. Here, we use a breast cancer preclinical model to identify resistance mechanisms to a small molecule allosteric AKT inhibitor, MK2206. Using a step-wise and chronic high-dose exposure, breast cancer cell lines harboring oncogenic PI3K resistant to MK2206 were established. Using this model, we reveal that AKT3 expression is markedly upregulated in AKT inhibitor-resistant cells. Induction of AKT3 is regulated epigenetically by the bromodomain and extra terminal domain proteins. Importantly, knockdown of AKT3, but not AKT1 or AKT2, in resistant cells restores sensitivity to MK2206. AKT inhibitor-resistant cells also display an epithelial to mesenchymal transition phenotype as assessed by alterations in the levels of E-Cadherin, N-Cadherin, and vimentin, as well as enhanced invasiveness of tumor spheroids. Notably, the invasive morphology of resistant spheroids is diminished upon AKT3 depletion. We also show that resistance to MK2206 is reversible because upon drug removal resistant cells regain sensitivity to AKT inhibition, accompanied by reexpression of epithelial markers and reduction of AKT3 expression, implying that epigenetic reprogramming contributes to acquisition of resistance. These findings provide a rationale for developing therapeutics targeting AKT3 to circumvent acquired resistance in breast cancer. Mol Cancer Ther; 15(8); 1964-74. ©2016 AACR.

Density and distribution of connexin 43 in corpus cavernosum tissue from diabetic and hypogonadal patients with erectile dysfunction.

AIM: Altered expression of connexin 43 (Cx43) has been postulated to be involved in the development and progression of various diseases including erectile dysfunction (ED). The aim of this study was to determine whether distribution and density of the gap junction protein Cx43 are altered in human corpus cavernosum (HCC) tissue samples derived from diabetic or hypogonadal patients with ED compared to those from normal subjects. METHODS: HCC tissue sections derived from normal, diabetic and hypogonadal subjects were fixed in 4% formaldehyde, embedded in paraffin and immunostained with a monoclonal mouse anti-rat Cx43 antibody. Cx43 density was expressed as the cumulative number of gap junction plaques per unit area of tissue corrected for number of 4',6-diamidino-2-phenylindole, dihydrochloride-labeled smooth muscle cells (dots per unit area corrected for number of cells). RESULTS: The distribution of Cx43 plaques in smooth muscle was not affected in tissues derived from diabetic or hypogonadal subjects with ED compared with those from normal subjects. However, the number of Cx43 plaques was significantly reduced in HCC tissues derived from diabetic or hypogonadal subjects (73 ± 8% and 68 ± 11% of normal, respectively), indicating reduced Cx43 gap junctions in diabetic and hypogonadal subjects with ED. CONCLUSIONS: Cx43 density in the HCC was diminished in tissue samples derived from diabetic or hypogonadal patients with ED compared to tissue samples from normal non-diabetic subjects. This marked decrease in Cx43 gap junction channels may contribute to attenuated gap junction function and to diminished erectile physiology.