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.

Background/Aim: 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. Materials and Methods: A breast cancer genomics data set (METABRIC, n=2509) was downloaded and analyzed, as was the effect c-Met and PKCλ inhibitors on ALDH1 high cell viability and tumor-sphere formation. Results: c-Met expression correlates with expression of PKCλ in breast cancer. Stage III-IV breast cancer patients with c-Met high PKCλ high ALDH1A3 high have a poorer prognosis than patients with c-Met low PKCλ low ALDH1A3 low. Foretinib and auranofin suppressed cell viability and tumor-sphere formation by ALDH1 high cells. These results suggest that c-Met and PKCλ are cooperatively involved in cancer progression and contribute to poor prognoses in breast cancer. Conclusion: c-Met and PKCλ are potentially useful prognostic markers and therapeutic targets in late-stage breast cancer. Breast cancer is the second most frequently diagnosed cancer worldwide, and the most commonly occurring cancer among women, with 2.09 million new cases (24.2% of all cancers in women) and 0.6 million cancer-related deaths annually (1). Breast cancers are classified based on their gene expression pattern (PAM 50) into at least six subtypes, including normallike, luminal A, luminal B, HER2-enriched, claudin-low and basal-like (2-5). Among these, basal-like breast cancers have stem-like properties and a poor prognosis (4). Nonetheless, the prognosis for breast cancer patients is good overall, though it is significantly poorer for patients with late-stage tumors (stage III or IV) (6). This is in large part because late-stage breast cancers are often resistant to standard medical treatments, such as conventional surgery, chemotherapy, and radiotherapy, which is reflected in their recurrence and metastasis (6). Consequently, the new pharmacological approach to managing late-stage breast cancers is greatly needed. Tumors are composed of populations of cancer cells and distinct cancer stem cells (CSCs), which are largely undifferentiated tumorigenic cells that exhibit such stem-like properties as self-renewal and multipotency (7, 8). Most CSCs are resistant to conventional chemo-and radiotherapies, and the development of targeted therapies against CSCs is very much needed to improve clinical outcomes. CSCs within breast tumors can be identified based on their expression of CD44, CD24 and aldehyde dehydrogenase 1 (ALDH1) (9, 10). ALDH1 is an enzyme that converts aldehydes to carboxylic acids and is abundant in normal stem/progenitor cells, and various CSCs, including those in breast cancers (9, 11). Among the ALDH1 gene family, ALDH1A1 and ALDH1A3 are known to be CSC markers in several cancers (12-17). In particular, ALDH1A3 reportedly contributes significantly to the ALDH1 activity detected in breast cancer cells, and its expression correlates significantly with cancer...

Background/Aim: SLC20A1 has been identified as a prognostic marker in ER+ breast cancer. However, the role of SLC20A1 expression in breast cancer subtypes other than the ER+ types remains unclear. Materials and Methods: Genomics datasets were downloaded and analyzed, and the effect of SLC20A1 knockdown using targeted siRNA on cell viability and tumor-sphere formation was assessed. Results: SLC20A1 high patients with ER+, claudin-low or basal-like breast cancers showed poor prognoses. SLC20A1 high patients treated with radiotherapy had poor clinical outcomes. SLC20A1 knockdown suppressed the viability of MDA-MB 231 (claudin-low), MDA-MB 468 (basal-like) and MCF-7 (ER+) cells, and tumor-sphere formation by ALDH1 high cells. These results suggest that SLC20A1 is involved in cancer progression and contributes to clinical outcomes in patients with ER+, claudin-low and basal-like breast cancers. Conclusion: SLC20A1 is a potential prognostic marker and therapeutic target in ER+, claudin-low and basal-like breast cancers.Breast cancer is the most commonly occurring cancer among women worldwide, with 2.1 million new cases (24.2% of all cancers in women) and 0.6 million cancer-related deaths (15.0% of all cancer-related deaths among woman) annually (1). Breast cancer is classified using immunohistochemistry (IHC) and gene expression patterns (PAM50) (2-7). Based on IHC, breast cancer is classified into four types: ER+ and/or PgR+ HER2-type, ER+ and/or PgR+ HER2+ type, ER-and PgR-HER2+ type and triple negative type (TNBC). Based on PAM50, breast cancer is classified into at least six subtypes: normal-like, luminal A, luminal B, HER2-enriched, claudin-low and basal-like (2-7). Among these, the luminal A and luminal B types express ER (8, 9), and some luminal B and HER2-enriched types express HER2 (4, 8-11). Many claudin-low and basal-like types overlap with the TNBC type (6,(12)(13)(14).Breast cancer treatment mainly entails surgery, radiotherapy and drug therapy, which may include chemotherapy, endocrine therapy and/or molecular target therapy. Overall, breast cancer prognosis is good. Endocrine therapy is selected against ER+ type and a HER2-targeted antibody, such as trastuzumab, is used to treat HER2 type (11,15,16). However, there is no effective drug or molecular targeted therapy for TNBC or its overlapping claudin-low and basal-like types. Consequently, those patients are treated only with surgery, radiotherapy and chemotherapy, and have poor prognoses (6,13,(15)(16)(17). Moreover, it is also known that in some of these cases, chemo-and/or radiotherapy actually stimulates cancer progression (18)(19)(20). It is therefore essential to identify effective prognostic markers and molecular targets that can be exploited for the treatment of the claudin-low and basallike breast cancer subtypes.A major hurdle that must be overcome for therapy to be effective against the claudin-low and basal-like subtypes is 43 This article is freely accessible online.

A cold-adapted protease subtilisin was successfully isolated by evolutionary engineering based on sequential in vitro random mutagenesis and an improved method of screening (H. Kano, S. Taguchi, and H. Momose, Appl. Microbiol. Biotechnol. 47:46–51, 1997). The mutant subtilisin, termed m-63, exhibited a catalytic efficiency (expressed as thek cat/Km value) 100% higher than that of the wild type at 10°C whenN-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide was used as a synthetic substrate. This cold adaptation was achieved with three mutations, Val to Ile at position 72 (V72I), Ala to Thr at position 92 (A92T), and Gly to Asp at position 131 (G131D), and it was found that an increase in substrate affinity (i.e., a decreasedKm value) was mostly responsible for the increased activity. Analysis of kinetic parameters revealed that the V72I mutation contributed negatively to the activity but that the other two mutations, A92T and G131D, overcame the negative contribution to confer the 100% increase in activity. Besides suppression of the activity-negative mutation (V72I) by A92T and G131D, suppression of structural stability was observed in measurements of activity retention at 60°C and circular dichroism spectra at 10°C.

A new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which we developed for the screening of mutant subtilisins with increased activity at low temperature. The m-51 mutant showed 70% higher catalytic efficiency, expressed by the k(cat)/K(m) value, than the wild-type at 10 degrees C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate. This cold-adaptation was achieved mainly by the increase in the k(cat) value in a temperature-dependent manner. Genetic analysis revealed that m-51 had three mutations, Ala-->Thr at position -31 (A-31T) in the prodomain, Ala-->Val at position 88 (A88V), and Ala-->Thr at position 98 (A98T). From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V. The A-31T mutation severely constrained the autoprocessing-mediated maturation of the pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis. No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51. In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points. In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred. This mutual effect may be related to the enhanced activity of the double mutant.