Glyoxalase 1 (GLO1) is a ubiquitous enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis that induces apoptosis. In this study, we found that GLO1 gene expression correlates with neoplasm histologic grade (χ2 test, p = 0.002) and is elevated in human basal-like breast cancer tissues. Approximately 90% of basal-like cancers were grade 3 tumors highly expressing both GLO1 and the cancer stem cell marker ALDH1A3. ALDH1high cells derived from the MDA-MB 157 and MDA-MB 468 human basal-like breast cancer cell lines showed elevated GLO1 activity. GLO1 inhibition using TLSC702 suppressed ALDH1high cell viability as well as the formation of tumor-spheres by ALDH1high cells. GLO1 knockdown using specific siRNAs also suppressed ALDH1high cell viability, and both TLSC702 and GLO1 siRNA induced apoptosis in ALDH1high cells. These results suggest GLO1 is essential for the survival of ALDH1-positive breast cancer stem cells. We therefore conclude that GLO1 is a potential therapeutic target for treatment of basal-like breast cancers.

c-Met is a receptor-type tyrosine kinase, which is involved in a wide range of cellular responses such as proliferation, motility, migration and invasion. It has been reported to be overexpressed in various cancers. However, the role of c-Met in breast cancer stem cells (CSCs) still remains unclear. We herein, show that c-Met expression is significantly elevated in Basal-like type of breast cancer in comparison with other subtypes. High expression of c-Met strongly correlated with the expression of two CSC markers, ALDH1A3 and CD133 in breast cancers. In addition, breast cancers at tumor stage III-IV expressing both c-Methigh and ALDH1A3high had poor prognosis. Furthermore, treatment with c-Met inhibitors (Crizotinib, Foretinib, PHA-665752 and Tivantinib) in MDA-MB157 cells with high c-Met protein expression resulted in significant suppression in cell viability, contrary to MDA-MB468 cells with low c-Met protein expression. These c-Met inhibitors also suppressed cell viability and tumor-sphere formation of ALDH1high breast cancer cells with high c-Met expression. These results suggest that c-Met in ALDH1 positive CSCs seems to play an important role in breast cancer repopulation. Therefore, we conclude that c-Met is a potential therapeutic target in ALDH1 positive breast CSCs.

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.

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Background/Aim: 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. Materials and Methods: 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 (ALDH1 high ). Results: Patients with normal-like, luminal A or luminal B breast cancer with p62 high had poor prognosis. Furthermore, patients with p62 high ALDH1A3 high luminal B type also exhibited poor prognoses. Knockdown of p62 suppressed viability and tumor-sphere formation by ALDH1 high cells of the luminal B-type cell lines BT-474 and MDA-MB- These results suggest that p62 is essential for cancerous progression of ALDH1-positive luminal B breast CSCs, and contributes to poor prognosis of luminal B breast cancer. Conclusion: p62 is potentially a prognostic marker and therapeutic target for ALDH1-positive luminal B breast CSCs.Breast cancer has the highest prevalence among cancers of women worldwide, with 2.26 million new cases (24.5% of all cancer cases in women) and 685,000 cancer-associated mortalities (15.5% of all cancer-associated mortalities among women) annually (1). Breast cancer is classified using two parameters: Immunohistochemistry and gene-expression patterns [prediction analysis of microarray 50 (PAM50)] (2-8). Based on its PAM50, breast cancer is classified into at least six subtypes: Normal-like, luminal A, luminal B, human epidermal growth factor receptor type 2 (HER2)-enriched, claudin-low and basal-like (5, 7, 8). Among these, the luminal B type expresses estrogen receptor, and certain luminal B tumors express HER2 and highly express proliferation-related genes such as marker of proliferation Ki-67 (MKI67). In addition, the luminal B type has poorer prognosis (7,(9)(10)(11)(12)(13)(14)(15). Breast cancer treatment mainly entails surgery, radiotherapy and drug therapy, including chemotherapy, endocrine therapy and molecular targeted therapy. However, there are still numerous 3299

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 ALDH1 high cells derived from triple negative breast cancer (TNBC) and basal-like breast cancer were examined. Results: GLO 1 high , PKCλ high , and ALDH1A3 high 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 ALDH1 high cells. TLSC702 also effectively inhibited tumorsphere formation and induced death in PKCλ knockout ALDH1 high 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.Breast cancer is the second most frequently diagnosed cancer worldwide (1). There are approximately 2.26 million new cases of breast cancer, which account for 24.5% of all cases of cancer in women (Global Cancer Statistics 2020) (2). In addition, breast cancer is responsible for approximately 680,000 cancer-associated deaths annually (2). Breast cancer is typically classified based on its receptor status and specific gene expression signature (PAM50) (3,4). In terms of receptor status, breast cancer is categorized into the estrogen receptor (ER)-positive, progesterone receptor (PgR)-positive, human epidermal growth factor receptor 2 (HER2)-positive or triple-negative breast cancer (TNBC; which is negative for ER, PgR, and HER2) subtypes (5,6). TNBC has the poorest prognosis among the four types of breast cancers, which is most likely due to its stem-like properties (7). Using PAM50 gene expression analysis, breast cancers can also be classified into ≥6 subtypes, namely normal-like, luminal A, luminal B, HER2enriched, claudin-low, and basal-like (3, 4). Among these subtypes, basal-like breast cancer is associated with poorer clinical outcomes, which is also at least partially due to its stem-like properties (3,8,9). Therefore, basal-like breast cancer has been frequently found to be either resistant or less responsive to conventional therapeutic approaches, including conventional surgery, chemotherapy and radiotherapy, resulting in high rates of recurrence and metastasis (10). In particular, 70-80% of basal-like breast cancers have also been reported to be of the TNBC subtype (11). Therefore, novel therapeutic targets for the effective treatment of TNBC and basal-like breast cancers are in demand. 5959This article is freely accessible online.