Expression of the CD44 gene is upregulated in breast cancer cells and is correlated with patient survival. Aberrant CD44 expression promotes tumor progression and metastasis. In the present study, we investigated the role of zerumbone (ZER) on regulatory mechanisms of CD44 expression in breast cancer cells. Our results showed that CD44 expression was significantly increased by epidermal growth factor receptor (EGFR) ligands in SKBR3 breast cancer cells. In contrast, EGF-induced CD44 expression was decreased by a MEK1/2 inhibitor, UO126, or STAT3 inhibitor, STAT3 VI, respectively. Notably, ZER downregulated the basal level of CD44 expression in CD44+ breast cancer cells. In addition, the induction of CD44 expression by EGFR ligands, EGF or TGF-α, was markedly decreased by ZER treatment. Finally, we investigated the inhibitory mechanism of ZER on EGF-induced CD44 expression. Our results showed that EGF-induced phosphorylation of STAT3 was completely suppressed by ZER. Collectively, ZER suppressed EGF-induced CD44 expression through inhibition of the STAT3 pathway. Therefore, we suggested that ZER may act as a promising therapeutic drug for the treatment of breast cancer.

Stanniocalcin‑1 (STC‑1), a secreted glycoprotein, is highly expressed in a variety of human malignancies. However, the role of STC‑1 has not been fully elucidated in breast cancer cells. Here, we investigated whether STC‑1 acts as a prognostic factor in triple‑negative breast cancer (TNBC) patients, and we explored the cellular mechanism in breast cancer cells. The level of STC‑1 expression was directly associated with the relapse‑free and overall survival of basal‑type breast cancer patients. Breast cancer patients with a high level of STC‑1 had poor prognosis. In addition, our results showed that the level of STC‑1 expression was markedly higher in TNBC than in non‑TNBC cells. Invasiveness of the TNBC cells was also significantly increased in response to recombinant human STC‑1 treatment. In contrast, the invaded cell numbers were completely decreased by STC‑1 siRNA overexpression in the Hs578T and MDA‑MB‑231 TNBC cells. Our results showed that the phosphorylation of c‑Jun N‑terminal protein kinase (JNK) and c‑Jun was increased after STC‑1 treatment but not the phosphorylation of ERK and p38 MAPKs in the Hs578T and MDA‑MB‑231 TNBC cells. Furthermore, expression of one invasion‑related gene MMP‑9, was increased by STC‑1 treatment. STC‑1‑induced MMP‑9 expression was suppressed by SP600125 (a JNK inhibitor) in the Hs578T cells. STC‑1‑induced cell invasiveness was also inhibited by SP600125. Taken together, we demonstrated that aberrant STC‑1 expression is associated with poor prognosis and stimulates the invasiveness of TNBC cells through the JNK/c‑Jun‑dependent signaling pathway.

Background/Aims: Transforming growth factor-beta proteins (TGF-βs) are multifunctional growth factors and powerful modulators of the epithelial-mesenchymal transition (EMT) in a variety of cancer types including breast and lung cancer cells. Here, we demonstrated the inhibitory effect of berberine (BBR) on tumor growth and metastasis of triple negative breast cancer (TNBC) cells via suppression of TGF-β1 expression. Methods: The levels of mRNA expression were analyzed by real-time PCR. The levels of MMP-2, MMP-9 and TGF-β1 protein expression were analyzed by zymography and confocal microscopy, respectively. Cell migration was analyzed by wound healing assay. Tumorigenicity of TNBC cells such as tumor growth and metastasis was analyzed using xenograft models. Results: In a clinical data set, aberrant TGF-β1 expression was associated with poor prognosis of breast cancer patients. Our in vitro results using TNBC cells showed that the expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 and the capacity for cell migration were increased by TGF-β1 treatment. In contrast, basal levels of MMP-2 and MMP-9 were suppressed by a specific TGF-β receptor I inhibitor, SB431542. In addition, TGF-β1–induced MMP-2 and MMP-9 expression and cell migration were decreased by SB431542. Interestingly, we showed for the first time that BBR decreased the level of TGF-β1, but not TGF-β2, in TNBC cells. Furthermore, BBR significantly decreased the level of MMP-2 expression as well as the capacity for cell migration in TNBC cells. Finally, we examined the effect of BBR on in vivo tumor growth and lung metastasis in MDA-MB231 and 4T1 breast cancer xenograft models and showed that both were significantly decreased following BBR treatment. Conclusion: BBR suppresses tumorigenicity of TNBC cells through inhibition of TGF-β1 expression. Therefore, we demonstrate that BBR could be a promising drug for treatment of TNBC.

Aberrant transforming growth factor-β (TGF-β) plays an important role in the development of cancer such as tumor metastasis and invasion. TGF-β-responsive gene signature is highly activated in chemotherapy-treated triple negative breast cancer (TNBC). Here, we investigated the effect of zerumbone (ZER) on TGF-β1 signaling pathway and tumorigenecity of TNBC cells. Our results showed that the level of TGF-β1 mRNA expression and cell invasiveness were higher in TNBC cells than in non-TNBC cells. On the other hand, the cell motility of TNBC cells was completely suppressed by LY2109761, a novel selective TGF-β receptor type I/II (TβRI/II) dual inhibitor. In addition, FN and MMP-2 expression, which play an important role on cell motility in various cancer cells, were dose-dependently decreased by LY2109761. TGF-β1 increased FN, MMP-2 and MMP-9 expression in HCC1806 TNBC cells. TGF-β1-induced MMP-9 expression was decreased by both a MEK inhibitor, UO126, and a smad3 inhibitor, SIS3. Induction of FN and MMP-2 by TGF-β1 was just decreased by SIS3. Overexpression of smad3 significantly increased FN, MMP-2, and MMP-9 expression. Interestingly, ZER significantly suppressed TGF-β1-induced FN, MMP-2, and MMP-9 expression in HCC1806 cells. In addition, ZER completely decreased TGF-β1-induced the phosphorylation of smad3. Finally, we observed that ZER suppressed the tumorigenecity such as tumor volume, weight, Ki67 expression, and metastasis in TNBC cells xenograft models. Taken together, we demonstrated that ZER suppresses TGF-β1-induced FN, MMP-2, and MMP-9 expression through the inactivation of smad3 and inhibits the tumorigenecity of TNBC cells. Therefore, we suggest that ZER may act as a promising drug for treatment of TNBC.