Research ArticleExperimental Studies

High Expression of c-Met, PKCλ and ALDH1A3 Predicts a Poor Prognosis in Late-stage Breast Cancer

HITOMI MOTOMURA, YUKA NOZAKI, CHOTARO ONAGA, AYAKA OZAKI, SHOMA TAMORI, TAKA-AKI SHIINA, SHOTARO KANAI, CHIHIRO OHIRA, YASUSHI HARA, YOHSUKE HARADA, RYOKO TAKASAWA, TAKEHISA HANAWA, SEI-ICHI TANUMA, YASUNARI MANO, TSUGUMICHI SATO, KEIKO SATO and KAZUNORI AKIMOTO
Anticancer Research January 2020, 40 (1) 35-52; DOI: https://doi.org/10.21873/anticanres.13924

Abstract

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 ALDH1high 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-Methigh PKCλhigh ALDH1A3high have a poorer prognosis than patients with c-Metlow PKCλlow ALDH1A3low. Foretinib and auranofin suppressed cell viability and tumor-sphere formation by ALDH1high 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 normal-like, 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 type, tumor grade and metastasis in breast cancer patients (16-19).

c-Met is a receptor-type tyrosine kinase that is involved in a wide range of cellular functions, including proliferation, motility, migration, invasion and tumor angiogenesis (20, 21). c-Met is reported to be highly expressed and aberrantly activated in a variety of cancers, including breast cancer (20-22). Moreover, high c-Met expression reportedly correlates with expression of the CSC markers CD133, CD44, and ALDH1 (18, 23-25), and appears to be involved in various processes in breast and head neck CSCs (18, 25). c-Met is enriched in late-stage basal-like breast cancers, which exhibit stem cell properties and strongly express ALDH1A3 (18). High c-Met and ALDH1A3 expression in late-stage breast tumors is predictive of a poor clinical outcome (18), but the relationship between c-Met and other signaling molecules in ALDH1-positive breast CSCs remains unclear.

PKCλ is one of the atypical protein kinase C (aPKC) subfamily proteins and is known to be involved in cellular processes contributing to cell polarity, proliferation, survival, chemotaxis, and migration (26-28). PKCλ is overexpressed in many human cancers, including breast cancer (29-40), where it is known to be involved in cancerous progression and to contribute to a poor clinical outcome (33-40). In ovarian cancer, lung cancer and glioblastoma, PKCλ is reportedly involved in cell proliferation and tumor formation by stem-like cells (41-44). However, the role of PKCλ in ALDH1-positive breast CSCs remains to be elucidated. In the present study, therefore, we investigated the relations among c-Met, PKCλ and ALDH1A3 in breast cancer. Our findings suggest that c-Met and PKCλ are potentially useful prognostic markers and therapeutic targets in late-stage breast cancers.

Materials and Methods

Analysis of cancer genomics data. Gene expression data were downloaded from the cBioportal and analyzed as previously described (18, 19, 45). Briefly, the Molecular Taxonomy of Breast Cancer International Consortium dataset (METABRIC, n=2509) and The Cancer Genome Atlas (TCGA) provisional datasets for several cancers were downloaded from cBioPortal (46, 47). For breast cancer, we classified tumor stage as early stage (Tumor stage 0-II) and late stage (Tumor stage III-IV). We defined the optimal cutoffs for the high and low expression groups using receiver operator characteristic (ROC) curves based on the expression of genes versus patient overall survival status. Values of p less than 0.05 were considered significant. Survival curves were plotted using the Kaplan-Meier method. For breast cancer, p-values were calculated using the Gehan-Breslow generalized Wilcoxon test; for other cancers, p-values were calculated using the log-rank test. A multivariate Cox regression model was used to evaluate the influence of gene expression and to estimate adjusted hazard ratios by age as a confounding factor. Values of copy-number alterations are shown as 2=high level amplification; 1=gain; 0=neutral/no change; −1=hemizygous deletion; −2=homozygous deletion. Statistical analysis was performed using BellCurve for Excel ver 2.11. (SSRI, Tokyo, Japan).

Cell culture. The MDA-MB 157 and MDA-MB 468 human basal-like breast cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were then cultured as previously described (18, 19).

Inhibitory compounds. Foretinib (c-Met inhibitor) was purchased from Selleck (Tokyo, Japan) and dissolved in dimethyl sulfoxide (DMSO). Auranofin (PKCλ inhibitor) was purchased from Santa Cruz Inc. (Dallas, TX, USA) and dissolved in DMSO.

ALDEFLUOR assay. ALDH1high cells were isolated from MDA-MB 157 cells using an ALDEFLUOR assay kit (Stem Cell Technology, Vancouver, BC, Canada) according to the manufacturer's instructions. As a negative control for the ALDEFLUOR assay, cells were incubated with the ALDH1 inhibitor diethylaminobenzaldehyde (DEAB). Approximately 5-10% of the total were sorted as ALDH1high cells by the cell sorter (FACS Aria III, BD Biosciences, San Jose, CA, USA), taking the negative control into consideration.

WST-8 assay. WST-8 assays were performed as previously described (18, 19). Briefly, cells (1×103/well) were seeded into 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 24 h. Inhibitors were then added to the culture medium, and the cells were incubated for an additional 7 days, after which cell viability was assessed using Cell Count Reagent SF (Nacalai tesque, Kyoto, Japan). The formazan dye formed was measured using a Sunrise Remote (TECAN, Männedorf, Switzerland) at 450 nm. Numerical values of the test groups are expressed as relative to cells in the absence of drug. Statistical significance was determined using Tukey's test. Data are presented as mean±SE of three independent experiments.

Tumor-sphere culture. Tumor-spheres were grown as previously described (18, 19). Briefly, cells (1×103/well) were seeded into 96-well ultralow attachment plates (Greiner Bio-One, Kremsmünster, Austria) and incubated for 24 h. Cells were cultured for 6 days while being treated with inhibitors. Images were taken through an inverted microscope (DMIL LED, Leica, Wetzlar, Germany), and the areas of tumor-spheres were determined using ImageJ software. Statistical significance was determined using Tukey's test. The areas of tumor-spheres are presented as mean±SE of three independent experiments. The number of tumor-spheres are presented as mean±SD of three independent experiments.

Immunoblot. Immunoblotting was performed as previously described (18, 19). Mouse anti-PKCι (λ) mAb was purchased from BD Biosciences (San Jose, CA, USA). Rabbit anti-c-Met pAb was from Santa Cruz Inc. (Dallas, TX, USA). Rabbit anti-phospho-Met (Tyr1234/1235) mAb was from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-ALDH1A3 pAb was from Invitrogen (Waltham, MA, USA). Mouse anti-β-actin mAb was from Proteintech Group, Inc. (Rosemont, IL, USA). Goat anti-mouse IgG and goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugate were from Cell Signaling Technology (Danvers, MA, USA).

Results

c-Met correlates with PKCλ in breast cancer. We previously reported that c-Met is elevated in basal-like breast cancers (18), while PKCλ was shown to be overexpressed in triple-negative (ER-negative, PgR-negative, HER2-negative) breast cancer (TNBC) (31). Approximately, 70-80% of basal-like breast cancers fall into the TNBC category (48). We therefore examined the correlation between c-Met and PKCλ expression in breast cancer. We found that c-Met expression correlated with expression of PKCλ in all patients (Figure 1A) and in several breast cancer subtypes, including normal-like, luminal B, claudin-low, and basal-like (Figure 1B). Our earlier report showed that in basal-like breast cancers, c-Met expression is higher at later tumor stages (stage III-IV) (18). In the present study, we found that the correlation between c-Met and PKCλ expression was stronger at late tumor stages than at early tumor stages (Figure 1A). On the other hand, PKCζ, another aPKC subfamily member, did not correlate with c-Met in all patients or for any breast cancer subtype (Figure 2A and B).

Figure 1.
Figure 1.

Correlation between c-Met and PKCλ expression in breast cancer. (A) Correlation between c-Met and PKCλ in all breast cancer patients at early tumor stages (0-II) and late tumor stages (III-IV). (B) Correlation between c-Met and PKCλ expression in the indicated breast cancer subtypes. Values are shown as scattered plots. Pearson's correlation coefficient (r) and p-value are indicated.

Late-stage breast cancer patients with c-Methigh PKCλhigh have a poorer prognosis. We next assessed the prognosis of patients with c-Methigh PKCλhigh (Figures 3 and 4). Kaplan-Meier analysis showed that patients with c-Methigh PKCλhigh had poorer clinical outcomes than those with c-Metlow PKCλlow for all tumors (Figure 3A, p=0.017), late-stage tumors (Figure 3C, p<0.01), and normal-like tumors (Figure 4, p=0.042). Multivariable Cox regression analysis also showed that c-Methigh PKCλhigh was predictive of a poorer prognosis than c-Metlow PKCλlow in all tumors (Table I, HR=1.25, 95%CI=1.04-1.51, p=0.017), late-stage tumors (Table I, HR=2.28, 95%CI=1.15-4.54, p=0.019), normal-like tumors (Table II, HR=2.45, 95%CI=1.10-5.48, p=0.029), and luminal A tumors (Table II, HR=1.72, 95%CI=1.14-2.60, p<0.01). These results suggest that c-Met and PKCλ act cooperatively towards cancer progression.

Figure 2.
Figure 2.

PKCζ expression does not correlate with c-Met expression in breast cancer. (A) Correlation between c-Met and PKCζ in all breast cancer patients at early tumor stages (0-II) and late tumor stages (III-IV). (B) Correlation between c-Met and PKCζ expression in the indicated breast cancer subtypes. Values are shown as scattered plots. Pearson's correlation coefficient (r) and p-value are indicated.

Late-stage breast cancer patients with c-Methigh PKCλhigh ALDH1A3high have a poorer prognosis. We have previously shown that late-stage breast cancer patients with c-Methigh ALDH1A3high have a poorer prognosis than those with c-Metlow ALDH1A3low (18). Here, we assessed the prognosis of patients with c-Methigh PKCλhigh ALDH1A3high (Figures 5 and 6). We found that late-stage patients with c-Methigh PKCλhigh ALDH1A3high had a poorer prognosis than those with c-Metlow PKCλlow ALDH1A3low (Figure 5C, p<0.01). Moreover, multivariate Cox regression analysis showed that c-Methigh PKCλhigh ALDH1A3high was predictive of a poorer prognosis than c-Metlow PKCλlow ALDH1A3low in all tumors (Table I, HR=1.55, 95%CI=1.21-1.99, p<0.01), early-stage tumors (Table I, HR=1.44, 95%CI=1.07-1.94, p=0.018), late-stage tumors (Table I, HR=2.47, 95%CI=1.08-5.65, p=0.031), and luminal A tumors (Table II, HR=1.79, 95%CI=1.07-2.99, p=0.026). These results suggest that c-Met and PKCλ act cooperatively towards the progression of ALDH1-positive breast CSCs.

Figure 3.
Figure 3.

High c-Met and PKCλ expression leads to poorer prognoses in late-stage cancers. Kaplan-Meier analysis of the effect of high c-Met and PKCλ expression on overall survival among all patients (A), Stage 0-II patients (B), and Stage III-IV patients (C). p-Value was calculated by the Gehan-Breslow generalized Wilcoxon test.

Auranofin suppresses ALDH1high cell viability and tumor-sphere growth. To examine the effects of c-Met and PKCλ co-expression in ALDH1-positive breast CSCs, we used the MDA-MB 157 human basal-like breast cancer cell line as a model. These cells express c-Met (18), phospho-Met (18), PKCλ and ALDH1A3 (19) (Figure 7A). ALDH1high cells derived from MDA-MB 157 exhibit such CSC-like properties as self-renewal, differentiation, and tumorigenesis (18). We have previously shown that the c-Met inhibitor foretinib suppresses cell viability and tumor-sphere formation by ALDH1high cells (18). In addition, the PKCλ inhibitor auranofin suppressed oncosphere formation and tumor growth by ovarian tumor-initiating cells (41). Consistent with these earlier observations, WST-8 assays showed that foretinib suppresses the cell viability of ALDH1high MDA-MB 157 cells (IC50=0.79 μM, Figure 7B), as did auranofin (IC50=0.24 μM, Figure 7C). However, the inhibitory combined effects of foretinib and auranofin on ALDH1high cell viability were neither additive nor synergistic as compared to the effect of each drug separately (Figure 7D).

Figure 4.
Figure 4.
Figure 4.

Kaplan-Meier analysis of the effect on overall survival of c-Met and PKCλ gene expression in the indicated breast cancer subtypes. p-Value was calculated by the Gehan-Breslow generalized Wilcoxon test.

To further assess the roles of c-Met and PKCλ in the tumorigenesis mediated by ALDH1-positive breast CSCs, we examined the effects of foretinib and auranofin on in vitro tumor-sphere formation. We found that foretinib and auranofin each suppressed tumor-sphere formation by ALDH1high cells (Figure 7E and F). As in the WST-8 assays, the combined inhibitory effects of foretinib and auranofin on tumor-sphere formation by ALDH1high cells were neither additive nor synergistic (Figure 7E and F). Moreover, the two drugs had no effect on tumor-sphere numbers when administered separately or together (Figure 7G). These results suggest that c-Met and PKCλ are cooperatively involved in ALDH1-positive breast CSC proliferation and tumor growth, acting via the same signaling pathway.

Discussion

Basal-like breast cancers are associated with poorer clinical outcomes than other breast cancer subtypes (4). c-Met mRNA is enriched in basal-like breast cancers as compared to other subtypes (18), while PKCλ is overexpressed in TNBC, which overlaps with basal-like breast cancers (31). We confirmed that in stage III-IV cancers, the fraction of basal-like breast cancer was significantly higher in c-Methigh PKCλhigh patients than in c-Metlow PKCλlow patients (Figure 8A). As shown in Figure 1B, the majority of basal-like breast cancer patients highly expressed c-Met and PKCλ (51.8%, n=103 of 199). Furthermore, the fraction of stage III-IV basal-like breast cancer patients with c-Methigh PKCλhigh ALDH1A3high was much larger than the fraction with c-Metlow PKCλlow ALDH1A3low patients (Figure 8B). In addition, late-stage patients with c-Methigh PKCλhigh ALDH1A3high had poorer prognoses than those with c-Metlow PKCλlow ALDH1A3low (Figure 5C). Taken together, these results strongly suggest that c-Met and PKCλ are cooperatively involved in cancer progression mediated by ALDH1-positive CSCs in late-stage basal-like breast cancer. The Kaplan-Meier analysis summarized in Table III indicates that cancer patients with c-Methigh PKCλhigh have poorer clinical outcomes. The patients' cancers included head and neck squamous cell carcinoma (p<0.01), pancreatic adenocarcinoma (p<0.01) and lung adenocarcinoma (p<0.01). Similarly, patients with c-Methigh PKCλhigh ALDH1A3high also had poorer clinical outcomes. In these cases, the patients' cancers included head and neck squamous cell carcinoma (p=0.026), cervical squamous cell carcinoma and endocervical adenocarcinoma (p=0.032), pancreatic adenocarcinoma (p<0.01), bladder urothelial carcinoma (p<0.01), lung adenocarcinoma (p=0.033) and ovarian serous cystadenocarcinoma (p<0.01) (Table III). These results suggest that the contributions of c-Met and PKCλ to the progression of ALDH1-positive CSCs in not limited to breast cancer, and that c-Met and PKCλ levels may be useful prognostic markers and therapeutic targets in a variety of cancers.

View this table:
Table I.

Multivariable Cox regression analysis of the association between c-Met, PKCλ and ALDH1A3 expression and breast cancer in all patients and patients with stage 0-II or III-IV cancers.

View this table:
Table II.

Multivariable Cox regression analysis of the association between c-Met, PKCλ and ALDH1A3 and the indicated breast cancer subtypes.

Figure 5.
Figure 5.

High c-Met, PKCλ and ALDH1A3 expression leads to poor prognoses in late-stage cancers. Kaplan-Meier analysis of the effect of high c-Met, PKCλ and ALDH1A3 expression on overall survival among all patients (A), Stage 0-II patients (B), and Stage III-IV patients (C). p-Value was calculated by the Gehan-Breslow generalized Wilcoxon test.

Figure 6.
Figure 6.
Figure 6.

Kaplan-Meier analysis of the effect on overall survival of c-Met, PKCλ and ALDH1A3 gene expression in the indicated breast cancer subtypes. p-Value was calculated by the Gehan-Breslow generalized Wilcoxon test.

In the present study, we showed that c-Methigh correlates with PKCλhigh in breast cancer (Figure 1) and that breast cancer patients with c-Methigh PKCλhigh have poorer clinical outcomes for all tumors and for late-stage tumors (Figure 3). c-Met gene amplification and mutations occur in cancers (49, 50). In breast cancers, however, amplification of c-Met copy number was low (Figure 9A and B). Likewise, we found that no c-Met missense, in-frame or truncation mutations occurred in breast cancers (Table IV). It thus appears that in breast cancer, higher c-Met mRNA expression and activation reflect higher transcription rather than gene amplification or mutation. By contrast, amplification and gain of c-Met copy number frequently occurs in glioblastoma multiforme (high level amplification; 2.8%, gain; 75.2%) and testicular germ cell cancer (gain; 64.7%) (Figure 9C). In addition, c-Met mutation and overexpression is seen in stage III-IV lung carcinoma (49). The c-Met mutation rate is less than 5% in other cancers (Table IV). It appears, therefore, that higher c-Met expression reflects increased transcription rather than gene amplification or mutation in various cancers and that it contributes to the progression of these cancers.

Hepatocyte growth factor (HGF), a c-Met ligand, enhances CXCR4 expression via PKCζ and promotes breast cancer invasion and metastasis (51). We found that expression of PKCζ does not correlate with c-Met expression in all patients and breast cancer subtypes (Figure 2). By contrast, c-Met correlated with PKCλ in all patients and breast cancer subtypes (Figure 1). These results suggest that PKCλ, but not PKCζ, is involved in c-Met-dependent cell proliferation and tumor formation, as well as other actions of ALDH1-positive breast CSCs. Remarkably, PKCλ controls the Notch signal pathway, a key driver of stemness in KRAS-mediated lung adenocarcinoma (LADC) (43) and in glioblastoma (44). In addition, PKCλ controls signaling by SOX2-hedgehog acyl transferase (HHAT), a master transcriptional regulator of stemness, in lung squamous cell carcinoma (42). Importantly, the stemness markers Notch1, Notch3, Sox2 and ALDH1A3 are all enriched in grade 3 tumors, which are a major component of basal-like breast cancers (19).

Figure 7.
Figure 7.

Foretinib and auranofin suppress cell viability and tumor-sphere formation by ALDH1high cells. (A) c-Met, phospho-Met, PKCλ and ALDH1A3 protein expression in MDA-MB 157 and MDA-MB 468 human basal-like breast cancer cell lines. β-actin was used as an internal control. (B) Cell viability of ALDH1high cells isolated from MDA-MB 157 cells treated for 7 days with foretinib (0.03, 0.1, 0.3, 1, 3 and 10 μM) was assessed in WST-8 assays. Values for the test groups are expressed relative to cells treated with 0.1% DMSO. (C) Cell viability of ALDH1high cells isolated from MDA-MB 157 cells treated for 7 days with auranofin (0.03, 0.1, 0.3, 1, 3 and 10 μM) was assessed using WST-8 assays. Values for the test groups are expressed relative to cells treated with 0.06% DMSO. (D) Cell viability of ALDH1high cells isolated from MDA-MB 157 cells treated for 7 days with or without foretinib (0.1, 0.3, 1 μM) and/or auranofin (0.1, 0.3, 1 μM) was assessed using WST-8 assays. Values for the test groups are expressed relative to cells treated with 0.01% DMSO. (E) Representative images of tumor-spheres formed by ALDH1high cells isolated from MDA-MB 157 cells treated for 6 days with foretinib and/or auranofin. Bars: 100 μm. (F), (G) Foretinib and/or auranofin suppressed tumor-sphere formation measured as tumor-sphere area (F) or number (G). Values for the test groups are expressed relative to cells treated with 0.01% DMSO. (B), (C), (D), (F): Data represent the mean±SE (three independent experiments). (G): Data represent the mean±SD (three independent experiments). (D), (F), (G): Statistical significance was determined by Tukey's test. p-Value (**p<0.01, *p<0.05); vs. 0.01% DMSO. N.S.: Not significant.

Figure 8.
Figure 8.

High fraction of basal-like breast cancers among breast cancer patients with c-Methigh PKCλhigh or c-Methigh PKCλhigh ALDH1A3high. (A) Fractions (%) of patients with the indicated breast cancer subtypes expressing c-Methigh PKCλhigh or c-Metlow PKCλlow in all patients and each tumor stage breast cancer. (B) Fractions (%) of patients with the indicated breast cancer subtypes expressing c-Methigh PKCλhigh ALDH1A3high or c-Metlow PKCλlow ALDH1A3low in all patients and each tumor stage breast cancer.

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Table III.

Patients with c-Methigh PKCλhigh or c-Methigh PKCλhigh ALDH1A3high have poorer prognoses in several cancers.

The inhibitory effects of foretinib and auranofin on ALDH1high cell viability and tumor-sphere formation were neither additive nor synergistic (Figure 7). This suggests that PKCλ is a major downstream mediator of c-Met signaling in ALDH1-positive breast CSCs. c-Met activation also leads to PI3-kinase activation, and the stimulatory effect of PKCλ on cell proliferation is PI3-kinase-dependent (52-54). In addition, PI3-kinase activates the small GTP protein rac (55). HGF/c-Met also mediated rac activation and breast cancer migration, while epithelial cell transforming sequence 2 (Ect2) interacts with the Par6-PKCλ complex to activate rac during cancer cell proliferation (56). This suggests the presence of a c-Met-PI3-kinase-Par6/PKCλ/Ect2-rac signaling axis in ALDH1-positive breast CSCs.

Conclusion

In summary, we showed that high c-Met expression correlates with high PKCλ expression in breast cancer. In addition, patients with late-stage tumors showing high expression of c-Met, PKCλ and ALDH1A3 had poorer prognoses. Treating ALDH1high cells with a c-Met or PKCλ inhibitor suppressed cell viability and tumor-sphere formation. These results suggest that c-Met and PKCλ in ALDH1-positive breast CSCs are involved in tumor progression and contribute to a poor prognosis in breast cancer. We therefore suggest that c-Met and PKCλ are useful prognostic markers and therapeutic targets in late-stage breast cancers.

View this table:
Table IV.

Fraction (%) of the indicated cancers exhibiting c-Met mutation.

Figure 9.
Figure 9.

Copy number status of c-Met expression in breast cancer and other cancers. (A) Copy number status of c-Met expression in all breast cancer patients and in patients with stage 0-II or stage III-IV tumors. (B) Copy number status of c-Met expression in patients with the indicated breast cancer subtypes. (C) Copy number status of c-Met expression in the indicated cancers. Values: 2=high level amplification; 1=gain, 0=neutral/no change, −1=hemizygous deletion, −2=homozygous deletion.

Acknowledgements

This work was supported by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2014-2018.

Footnotes

  • Authors' Contributions

    H.M., Y.N., C.O., A.O., S.T., T.S., S.K., C.O., and Y.H. performed the experiments; H.M., Y.N., C.O., A.O., S.T., and Y.H. analyzed the data; H.M., Y.N., C.O., S.T., Y.M., T.S., and K.S. performed bioinformatics; Y.H., R.T., T.H., and S.-I.T. supplied experimental materials and resources; H.M., Y.N., and K.A. conceived the study; H.M. drafted the manuscript; H.M., Y.N., and K.A. contributed to discuss and review the final manuscript; all Authors approved the final manuscript.

  • This article is freely accessible online.

  • Conflicts of Interest

    The Authors state that they have no conflicts of interest to declare in regard to this study.

  • Received November 13, 2019.
  • Revision received November 21, 2019.
  • Accepted November 25, 2019.

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High Expression of c-Met, PKCλ and ALDH1A3 Predicts a Poor Prognosis in Late-stage Breast Cancer
HITOMI MOTOMURA, YUKA NOZAKI, CHOTARO ONAGA, AYAKA OZAKI, SHOMA TAMORI, TAKA-AKI SHIINA, SHOTARO KANAI, CHIHIRO OHIRA, YASUSHI HARA, YOHSUKE HARADA, RYOKO TAKASAWA, TAKEHISA HANAWA, SEI-ICHI TANUMA, YASUNARI MANO, TSUGUMICHI SATO, KEIKO SATO, KAZUNORI AKIMOTO
Anticancer Research Jan 2020, 40 (1) 35-52; DOI: 10.21873/anticanres.13924
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