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BACKGROUND/AIM We examined the inhibitory effects of both glyoxalase 1 (GLO 1) and protein kinase C (PKC)λ in aldehyde dehydrogenase 1 (ALDH1)-positive breast cancer stem cells (CSCs). MATERIALS AND METHODS Breast cancer genomics datasets (TCGA, n=593; METABRIC, n=1904) were downloaded and statistically analyzed. The effects of GLO 1 and PKCλ on trypan blue staining and tumor-sphere formation by ALDH1high cells derived from triple negative breast cancer (TNBC) and basal-like breast cancer were examined. RESULTS GLO 1high, PKCλhigh, and ALDH1A3high tumors were enriched in stage I/II/III/IV samples, associated with the HER2 and TNBC subtypes according to receptor status, and associated with the HER2-enriched and basal-like subtypes according to PAM50. Inhibition of either GLO 1 (TLSC702) or PKCλ (ANF) suppressed tumor-sphere formation and enhanced death in ALDH1high cells. TLSC702 also effectively inhibited tumor-sphere formation and induced death in PKCλ knockout ALDH1high cells. CONCLUSION GLO 1 and PKCλ are important for the survival of ALDH1-positive breast CSCs, and may represent potential therapeutic targets for the treatment of ALDH1-positive breast CSCs.

CD58 is an immune adhesion molecule on the cellular surface. It was previously found that a high expression of CD58 predicted a poor prognosis of patients with lower-grade gliomas. Therefore, the aim of this paper was to investigate the association between CD58 and breast cancer.CD58 gene expression data downloaded from cBioPortal was compared between the different subtypes of breast cancer. Clinical prognosis was examined using Kaplan-Meier analysis and multivariable Cox regression analysis. The association between CD58 expression and immune cell infiltration was estimated using the TIMER 2.0 web platform. Finally, the tumour sphere formation of aldehyde dehydrogenase 1 (ALDH1)CD58 mRNA was mainly enriched in claudin-low and basal-like subtypes. The high expression of CD58 predicted a good prognosis in patients with luminal A and luminal B breast cancer. This prediction may be due to the association of immune cell infiltration with CD58. Notably, patients with luminal A breast cancer with a high expression of CD58 in association with ALDH1A3 exhibited a good prognosis; however, this did not apply to patients with basal-like breast cancer. The in vitro experiments revealed that knockdown of CD58 inhibited the tumour sphere formation ability of ALDH1CD58 may function as a potential prognostic biomarker and therapeutic target in ALDH-positive basal-like cancer stem cells.

p62 (also known as sequestosome 1) is involved in cancer progression, and high expression of p62 indicates poor clinical outcome in several cancer types. However, the association between p62 gene expression and cancer stem cells (CSCs) in breast cancer subtypes remains unclear.In the present study, genomic datasets of primary breast cancer (The Cancer Genome Atlas, n=593; and Molecular Taxonomy of Breast Cancer International Consortium, n=2,509) were downloaded. p62 Expression was then examined in normal and breast cancer tissues derived from the same patients. Kaplan-Meier and multivariate Cox regression analyses were employed to evaluate disease-specific survival. Next, the effect on cell viability and in vitro tumor-sphere formation of p62 knockdown using targeted small interfering RNA was assessed by using cells with high activity of aldehyde dehydrogenase 1 (ALDH1Patients with normal-like, luminal A or luminal B breast cancer with p62p62 is potentially a prognostic marker and therapeutic target for ALDH1-positive luminal B breast CSCs.

Estrogen receptor-positive (ER+) breast cancer intrinsically confers satisfactory clinical outcomes in response to endocrine therapy. However, a significant proportion of patients with ER+ breast cancer do not respond well to this treatment. Therefore, to evaluate the effects of endocrine therapy, there is a need for identification of novel markers that can be used at the time of diagnosis for predicting clinical outcomes, especially for early-stage and late recurrence. Solute carrier family 20 member 1 (SLC20A1) is a sodium/inorganic phosphate symporter that has been proposed to be a viable prognostic marker for the luminal A and luminal B types of ER+ breast cancer. In the present study, we examined the possible association of SLC20A1 expression with tumor staging, endocrine therapy and chemotherapy in the luminal A and luminal B subtypes of breast cancer. In addition, we analyzed the relationship between SLC20A1 expression and late recurrence in patients with luminal A and luminal B breast cancer following endocrine therapy. We showed that patients with higher levels of SLC20A1 expression (SLC20A1high) exhibited poorer clinical outcomes in those with tumor stage I luminal A breast cancer. In addition, this SLC20A1high subgroup of patients exhibited less responses to endocrine therapy, specifically in those with the luminal A and luminal B subtypes of breast cancer. However, patients with SLC20A1high showed good clinical outcomes following chemotherapy. Patients tested to be in the SLC20A1high group at the time of diagnosis also showed a higher incidence of recurrence compared with those with lower expression levels of SLC20A1, at >15 years for luminal A breast cancer and at 10–15 years for luminal B breast cancer. Therefore, we conclude that SLC20A1high can be used as a prognostic biomarker for predicting the efficacy of endocrine therapy and late recurrence for ER+ breast cancer.

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, χ2-test). Late stage breast cancer patients expressing PKCλhigh and ALDH1A3high had poorer disease-specific survival than those expressing PKCλlow and ALDH1A3low (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 ALDH1high 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 ALDH1high 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 ALDH1high cells. It also altered the asymmetric/symmetric distribution ratio of ALDH1A3 protein. In addition, PKCλ knockdown led to increases in cellular ROS levels in ALDH1high 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.

Co-expression of c-Met and ALDH1A3 indicates a poor prognosis in stage III-IV breast cancers and contributes to cell proliferation and tumor formation by ALDH1-positive breast CSCs. PKCλ is overexpressed and contributes to a poor prognosis in several cancers.A breast cancer genomics data set (METABRIC, n=2509) was downloaded and analyzed, as was the effect c-Met and PKCλ inhibitors on ALDH1c-Met expression correlates with expression of PKCλ in breast cancer. Stage III-IV breast cancer patients with c-Metc-Met and PKCλ are potentially useful prognostic markers and therapeutic targets in late-stage breast cancer.