High PKCλ expression is required for ALDH1-positive cancer stem cell function and indicates a poor clinical outcome in late-stage breast cancer patients

Abstract

Despite development of markers for identification of cancer stem cells, the mechanism underlying the survival and division of cancer stem cells in breast cancer remains unclear. Here we report that PKCλ expression was enriched in basal-like breast cancer, among breast cancer subtypes, and was correlated with ALDH1A3 expression (p = 0.016, χ 2-test). Late stage breast cancer patients expressing PKCλ high and ALDH1A3 high had poorer disease-specific survival than those expressing PKCλ low and ALDH1A3 low (p = 0.018, log rank test for Kaplan-Meier survival curves: hazard ratio 2.58, 95% CI 1.24-5.37, p = 0.011, multivariate Cox regression analysis). Functional inhibition of PKCλ through siRNA-mediated knockdown or CRISPR-Cas9-mediated knockout in ALDH1 high MDA-MB 157 and MDA-MB 468 basal-like breast cancer cells led to increases in the numbers of trypan blue-positive and active-caspase 3-positive cells, as well as suppression of tumor-sphere formation and cell migration. Furthermore, the amount of CASP3 and PARP mRNA and the level of cleaved caspase-3 protein were enhanced in PKCλ-deficient ALDH1 high cells. An Apoptosis inhibitor (z-VAD-FMK) suppressed the enhancement of cell death as well as the levels of cleaved caspase-3 protein in PKCλ deficient ALDH1 high cells. It also altered the asymmetric/symmetric distribution ratio of ALDH1A3 protein. In addition, PKCλ knockdown led to increases in cellular ROS levels in ALDH1 high cells. These results suggest that PKCλ is essential for cancer cell survival and migration, tumorigenesis, the asymmetric distribution of ALDH1A3 protein among cancer cells, and the maintenance of low ROS levels in ALDH1-positive breast cancer stem cells. This makes it a key contributor to the poorer prognosis seen in late-stage breast cancer patients.

Glyoxalase 1 and protein kinase Cλ as potential therapeutic targets for late‑stage breast cancer

“…Gene expression data was downloaded from cBioportal and analyzed as previously described (21,41,42,53,54). Briefly, the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset (55,56) was downloaded from cBioPortal (cbioportal.…”

“…MCF-10A cells were grown in mammary epithelial cell growth medium (MEGM; Lonza Group, Ltd.) according to instructions from ATCC. The cancer cell lines were cultured as previously described (21,41,42,53). Mycoplasma testing was performed on all the cell lines used.…”

“…Immunoblotting was performed as previously described (21,41,42,53). The primary antibodies used were: Mouse anti-PKCι mAb (1:5,000; cat.…”

“…One of the atypical protein kinase C (aPKC) isotypes, PKCλ/ι, is known to be involved in cellular responses that include determination of cell polarity, as well as cell proliferation, survival, chemotaxis and migration (24)(25)(26). PKCλ is overexpressed in several types of cancer, including breast cancer (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42), and is known to be involved in cancer progression, contributing to poor clinical outcomes (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). In TNBC cells, TGFβ and IL1β induce PKCλ phosphorylation and promote PKCλ-dependent proliferation, invasiveness and metastasis by inducing NF-κB p65 nuclear translocation (30).…”

“…c-Met and PKCλ are cooperatively involved in cellular viability and tumor formation in basal-like breast cancer cells (42). PKCλ is also essential for the survival of ALDH1-positive breast cancer stem cells in a caspase-3-dependent manner (41,42). EGF, platelet-derived growth factor and insulin collectively activate PKCλ via PI3-kinase (43)(44)(45), and PKCλ subsequently binds to and regulates p70 S6 kinase (46).…”