Search

Cluster of differentiation 133 (CD133) is a transmembrane glycoprotein that has been reported as a marker of cancer stem cells or cancer-initiating cells in various cancers. However, its contribution to tumorigenesis and differentiation remains to be elucidated. To determine the role of CD133 in colon cancer, we silenced CD133 in human colon cancer cells. Silencing of CD133 results in decreased cell proliferation, survival, migration, invasion, and glucose transport. These effects are mediated by downregulation of the human epidermal growth factor receptor 3 (HER3)/Akt/mTOR signaling pathway, culminating in reduced expression of the glucose transporter GLUT1. We also confirm that the cellular phenotypes of CD133-silenced cells are mediated by GLUT1 downregulation. We conclude that CD133 is a potential tumor initiator that positively regulates GLUT1 expression through modulation of HER3/Akt/mTOR signaling.

Members of the EGFR family are potent mediators of normal cell growth and development. HER2 possesses an active tyrosine kinase domain, but no direct ligand has been identified. To investigate the differential effect of HER2 in breast cell lines, HER2 was overexpressed in MCF-10A, MCF7 and MDA-MB-231 cells. HER2 overexpression promoted proliferation, survival and migration in MCF-10A and MCF-7 cells. No significant differences were seen in proliferation, survival or migration between MDA-MB-231 vec and HER2 cells. The activity of downstream HER2 proteins increased in MCF-10A HER2 and MCF-7 HER2 cells but not in MDA-MB-231 HER2 cells. Exogenously expressed HER2 failed to associate with EGFR or HER3 in MDA-MB-231 cells, while overexpression of HER2 enhanced HER family dimerization in MCF-10A and MCF-7 cells.

Purpose: Keratin19 (KRT19) is the smallest known type I intermediate filament and is used as a marker for reverse transcriptase PCR–mediated detection of disseminated tumors. In this study, we investigated the functional analysis of KRT19 in human breast cancer. Experimental Design: Using a short hairpin RNA system, we silenced KRT19 in breast cancer cells. KRT19 silencing was verified by Western blot analysis and immunocytochemistry. We further examined the effect of KRT19 silencing on breast cancer cells by cell proliferation, migration, invasion, colony formation assay, cell-cycle analysis, immunocytochemistry, immunohistochemistry, and mouse xenograft assay. Results: Silencing of KRT19 resulted in increased cell proliferation, migration, invasion, and survival. These effects were mediated by upregulation of Akt signaling as a result of reduced PTEN mRNA expression. Silencing of KRT19 decreased the nuclear import of early growth response-1 (Egr1), a transcriptional factor for PTEN transcription, through reduced association between Egr1 and importin-7. We also confirmed that silencing of KRT19 increased tumor formation in a xenograft model. Conclusions: KRT19 is a potential tumor suppressor that negatively regulates Akt signaling through modulation of Egr1 nuclear localization. Clin Cancer Res; 19(16); 4335–46. ©2013 AACR.

Cluster of differentiation 24 (CD24) is a small glycosylphosphatidylinositol-linked cell surface molecule that is expressed in a variety of human carcinomas, including breast cancer. To determine the role of CD24 in breast cancer cells, we expressed CD24 in CD24-negative/low and cluster of differentiation 44 (CD44)-positive MDA-MB-231 metastatic breast cancer cells. Forced expression of CD24 resulted in a decrease in c-Raf/mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase signaling and reduced cell proliferation. Apoptosis induced by DNA damage was greatly enhanced in MDA-MB-231 CD24 cells as compared with MDA-MB-231 vec cells. CD24 expression efficiently attenuated DNA damage-induced nuclear factor-kappaB (NF-κB) signaling in MDA-MB-231 cells. However, in CD24-positive and CD44-negative/low MCF-7 cells, knockdown of CD24 did not significantly affect DNA damage-induced apoptosis nor NF-κB signaling. Silencing of CD24 in CD24/CD44-double-positive MDA-MB-468 cells partially rescued DNA damage-induced apoptosis. Transient transfection studies with 293T cells also revealed that CD24 attenuated cell viability and NF-κB signaling only when CD44 was cotransfected. These data indicate that CD24 expression potentiated DNA-induced apoptosis by suppressing antiapoptotic NF-κB signaling in CD44-expressing cells.

In this study, we investigated the underlying mechanism by which phytoestrogens suppress the growth of normal (MCF-10A) and malignant (MDA-MB-231) estrogen receptor α (ERα)-negative breast cells. We hypothesized that phytoestrogen inhibits the proliferation of ERα-negative breast cancer cells. We found that all tested phytoestrogens (genistein, apigenin, and quercetin) suppressed the growth of both MCF-10A and MDA-MB-231 cells, as revealed by proliferation assays. These results were accompanied by an increase in the sub-G0/G1 apoptotic fractions as well as an increase in the cell population in the G2/M phase in both cell types, as revealed by cell cycle analysis. When we assessed the effect of phytoestrogens on the level of intracellular signaling molecules by Western blot analysis, we found that phytoestrogens increased the level of active p53 (phospho-p53) without changing the p53 level in both MCF-10A and MDA-MB-231 cells. Phytoestrogens also induced an increase in p21, a p53 target gene, and a decrease in either Bcl-xL or cyclin B1 in both cell types. In contrast, the protein levels of phosphatase and tensin homolog, cyclin D1, cell division control protein 2 homolog, phospho-cell division control protein 2 homolog, and p27 were not changed after phytoestrogen treatment. Our data indicate that phytoestrogens induce apoptotic cell death of ERα-negative breast cancer cells via p53-dependent pathway and suggest that phytoestrogens may be promising agents in the treatment and prevention of ERα-negative breast cancer.

To dissect the isoform-specific roles of Akt in breast cancer cells, constitutively active Akt isoforms were introduced into MDA-MB-231 cells. Both Akt1 and Akt2 efficiently inhibited the growth of MDA-MB-231 cells. Overexpression of Akt1 down-regulated ERK activity inhibiting Ser 259 phosphorylation of c-Raf and subsequent downstream signaling. Akt2 overexpression up-regulated the cell cycle inhibitor p27. Cycloheximide decay assays showed that Akt2 increased the stability and nuclear localization of p27, thus inhibiting the cyclin E/CDK2 complex. These results suggest that the inhibition of cell proliferation by Akt1 and Akt2 is mediated by isoform-specific mechanisms.

S100A4, also known as the mts1 gene, has been reported as an invasive and metastatic marker for many types of cancers. S100A4 interacts with various target genes that affect tumor cell metastasis; however, little is known about cellular signaling pathways elicited by S100A4. In the current study, we demonstrate an inhibitory effect of S100A4 on β-catenin signaling in breast cancer cells. By overexpressing S100A4 in MCF-7, MDA-MB-231 and MDA-MB-453 breast cancer cells, we observed the down-regulation of β-catenin expression and β-catenin-dependent TCF/LEF transcriptional activities. The activity of GSK3β, which phosphorylates β-catenin and induces proteasomal degradation of β-catenin, was increased in S100A4-overexpressing cell lines. Blocking Glycogen Synthase Kinase (GSK3β) activity by lithium chloride or Dvl gene overexpression restored β-catenin expression. We also found that increased GSK3β activity was due to decrease in Akt activity resulting from Egr-1-induced phosphatase and tensin homolog (PTEN) expression. S100A4 induced Egr-1 nuclear localization by increasing the association between Egr-1 and importin-7 and this effect was reduced in S100A4 mutants that harbored a defect in nuclear localization signals. Collectively, we verify herein that S100A4 may act as a tumor suppressor in breast cancers by down-regulating the central signaling axis for tumor cell survival.

In breast cancer, the HER2 (human epidermal growth factor receptor 2) receptor tyrosine kinase is associated with extremely poor prognosis and survival. Notch signalling has a key role in cell-fate decisions, especially in cancer-initiating cells. The Notch intracellular domain produced by Notch cleavage is translocated to the nucleus where it activates transcription of target genes. To determine the combinatory effect of HER2 and Notch signalling in breast cancer, we investigated the effect of HER2 on Notch-induced cellular phenomena. We found the down-regulation of Notch-dependent transcriptional activity by HER2 overexpression. Also, the HER2/ERK (extracellular-signal-regulated kinase) signal pathway down-regulated the activity of γ-secretase. When we examined the protein level of Notch target genes in HER2-overexpressing cells, we observed that the level of survivin, downstream of Notch, increased in HER2 cells. We found that activation of ERK resulted in a decrease in XAF1 [XIAP (X-linked inhibitor of apoptosis)-associated factor 1] which reduced the formation of the XIAP–XAF1 E3 ligase complex to ubiquitinate survivin. In addition, Thr34 of survivin was shown to be the most important residue in determining survivin stability upon phosphorylation after HER2/Akt/CDK1 (cyclin-dependent kinase 1)–cyclin B1 signalling. The results of the present study show the combinatorial effects of HER2 and Notch during breast oncogenesis.

Background The clinical implication of Ras/Raf/ERK pathway activity in breast cancer tissue and its association with response to chemotherapy is controversial. We aimed to explore the value of p90RSK phosphorylation, a downstram molecule of the pathway, in predicting chemotherapy response in breast cancer. Methods The expression of phosphorylated p90RSK (phospho-p90RSK) and chemotherapy response was measured in 11 breast cancer cell lines and 21 breast cancer tissues. The predictive value of phospho-p90RSK was validated in core needle biopsy specimens of 112 locally advanced breast cancer patients who received anthracycline and taxane-based neoadjuvant chemotherapy. Results In 11 breast cancer cell lines, the relative expression of phospho-p90RSK was inversely correlated with cell survival after doxorubicin treatment (p = 0.021). Similar association was observed in fresh tissues from 21 breast cancer patients in terms of clinical response. In paraffin-embedded, formalin-fixed tissues from core needle biopsy tissues from 112 patients, positive phospho-p90RSK expression was associated with greater tumor shrinkage and smaller post-chemotherapy tumor size. The association between phospho-p90RSK expression and chemotherapy response was more evident in estrogen receptor(ER)-positive tumors. The expression of phosphor-p90RSK did not show a significant relationship with the incidence of pCR. P90RSK silencing using siRNA did not affect the cancer cell’s response to doxorubicin, and the expression of phospho-p90RSK was highly correlated with other Ras/Raf/ERK pathway activation. Conclusion Our results suggest that phospho-p90RSK expression, which reflects the tumor’s Ras/Raf/ERK/p90RSK pathway activation can be a potential predictive marker for chemotherapy response in ER-positive breast cancer which needs further independent validation.

To determine the role of CD24 in breast cancer cells, we knocked down CD24 in MCF-7 human breast cancer cells by retroviral delivery of shRNA. MCF-7 cells with knocked down CD24 (MCF-7 hCD24 shRNA) exhibited decreased cell proliferation and cell adhesion as compared to control MCF-7 mCD24 shRNA cells. Decreased proliferation of MCF-7 hCD24 shRNA cells resulted from the inhibition of cell cycle progression from G1 to S phase. The specific inhibition of MEK/ERK signaling by CD24 ablation might be responsible for the inhibition of cell proliferation. Phosphorylation of Src/FAK and TGF-β1-mediated epithelial to mesenchymal transition was also down-regulated in MCF-7 hCD24 shRNA cells. Reduced Src/FAK activity was caused by a decrease in integrin β1 bound with CD24 and subsequent destabilization of integrin β1. Our results suggest that down-regulation of Raf/MEK/ERK signaling via Src/FAK may be dependent on integrin β1 function and that this mechanism is largely responsible for the CD24 ablation-induced decreases in cell proliferation and epithelial to mesenchymal transition.

We performed a phase II study of combination chemotherapy with S-1 plus gemcitabine for treating chemo-naïve patients with unresectable pancreatic cancer to evaluate the efficacy and toxicity.Patients with histologically confirmed unresectable pancreatic cancer were eligible. The treatment consisted of S-1 (40 mg/m(2) p.o. b.i.d. from D1 to 14) and gemcitabine (1,250 mg/m(2) on D1 and 8), repeated every 3 weeks.Thirty-two patients were enrolled between March 2005 and December 2007. No complete response was observed and a partial response was observed in 14 patients (44.0%), stable disease in eight patients (25.0%), and progressive disease in eight patients (25.0%). The median time to progression was 4.92 months (95% CI: 4.16-5.67 months), and the median overall survival was 7.89 months (95% CI: 5.96-9.82 months). The survival duration was significantly longer for the patients with a good performance status compared with that of the patients with a poor performance status. The major toxicities were grade 3-4 neutropenia (9, 28.1%), grade 3/4 thrombocytopenia (5, 15.6%), and grade 3 diarrhea (5, 15.6%).The combination chemotherapy of S-1 and gemcitabine showed promising antitumor activity and manageable toxicities, and especially for the good performance status patients with unresectable pancreatic cancer.