An automated epifluorescence microscopy imaging assay for the identification of phospho-AKT level modulators in breast cancer cells

Abstract

AKT is an enzyme of the PI3K/pAKT pathway, regulating proliferation and cell survival. High basal levels of active, phosphorylated AKT (pAKT) are associated with tumor progression and therapeutic resistance in some breast cancer subtypes, including HER2 positive breast cancers. Various stimuli can increase pAKT levels and elevated basal pAKT levels are a feature of PTEN-deficient breast cancer cell lines. The aim of this study was to develop an assay able to identify modulators of pAKT levels using an automated epifluorescence microscope and high content analysis. To develop this assay, we used HCC-1569, a PTEN-deficient, HER2-overexpressing breast cancer cell line with elevated basal pAKT levels. HCC-1569 cells were treated with a selective pharmacological inhibitor of AKT (MK-2206) to reduce basal pAKT levels or EGF to increase pAKT levels. Immunofluorescence images were acquired using an automated epifluorescence microscope and integrated intensity of cytoplasmic pAKT staining was calculated using high content analysis software. Mean and median integrated cytoplasmic intensity were normalized using fold change and standard score to assess assay quality and to identify most robust data analysis. The highest z' factor was achieved for median data normalization using the standard score method (z' = 0.45). Using our developed assay we identified the calcium homeostasis regulating proteins TPRV6, STIM1 and TRPC1 as modulators of pAKT levels in HCC-1569 cells. Calcium signaling controls a diverse array of cellular processes and some calcium homeostasis regulating proteins are involved in modulating pAKT levels in cancer cells. Thus, these identified hits present promising targets for further assessment.

The TRPC1 Channel Forms a PI3K/CaM Complex and Regulates Pancreatic Ductal Adenocarcinoma Cell Proliferation in a Ca2+-Independent Manner

“…We found that TRPC1 silencing inhibited 2D cell proliferation and spheroid growth. The same effect has been found in neuroblastoma [22], glioblastoma [23], thyroid [24], breast [12,[25][26][27], lung, [28], liver [29,30], ovarian [31], and colon cancer [13]. This indicates that the upregulation of TRPC1 stimulates proliferation in several cancer types.…”

“…However, it seems that it depends on the cancer cell type, and whether the presence or absence of TRPC1 contributes to cell growth. In a majority of studies, TRPC1 downregulation inhibits the proliferative rate in neuroblastoma [ 22 ], glioblastoma [ 23 ], thyroid [ 24 ], breast [ 12 , 25 , 26 , 27 ], lung, [ 28 ], liver [ 29 , 30 ], ovarian [ 31 ], and colon cancer [ 13 ]. Whereas in other studies, TRPC1 downregulation drives proliferation and growth in esophageal [ 32 ] and breast cancer [ 33 ].…”

Expression of TRPC1 and SBEM protein in breast cancer tissue and its relationship with clinicopathological features and prognosis of patients

“…TRPC1, as a potential target molecule for diagnosis and treatment of early breast cancer, is abnormally elevated in a variety of tumor tissues including breast cancer. Some research indicates that it can play a role in inducing apoptosis of breast cancer cells by inhibiting expression of apoptosis protein-related proteins (21,22). By constructing specific TRPC1 siRNA expression vector and lentivirus vector mediated TRPC1 siRNA interference system, transfecting cells and establishing stable passage cell lines, it was found that after down-regulation of TRPC1 gene, breast cancer cell proliferation, invasion and migration ability were down-regulated, and apoptosis rate of cancer cells increased (23,24).…”

Store-Operated Ca2+ Entry in Breast Cancer Cells: Remodeling and Functional Role

“…TRPC1 has also been reported to be involved in very specific aspects of EMT remodeling and induction [86]. The role of TRPC1 in the modulation of Akt phosphorylation has been confirmed by Kaemmerer et al in the ER - /HER2 + epithelial breast cancer HCC1569 cell line [85]. Consistent with a role for TRPC1 in EMT induction, silencing TRPC1 expression has been reported to impair the activation of EMT stimulated by TGFβ in the murine mammary epithelial NMuMG cell line; meanwhile, TRPC1 overexpression increased TGFβ-induced EMT in these cells [78], which strongly supports a role for TRPC1 in the induction of EMT in breast cells.…”