Atsuko Yamada

Extracellular signal-regulated kinase and Akt activation play a critical role in the process of hepatocyte growth factor-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) has recently been studied to elucidate mechanisms of the liver metastatic process. We investigated EMT in the process of liver metastasis and the effects of chemotherapy on EMT cells as therapeutic strategy for colorectal liver metastasis. We used the CT26 murine colorectal carcinoma cell line to create an in vivo mouse liver metastasis model. Liver tumors were stained immuno-histochemically. Expression of proteins associated with TGF-β/Smad and hepatocyte growth factor (HGF)/c-Met pathways were investigated by western blotting. Cells with c-Met mRNA knockdown by siRNA techniques showed clearly reduced liver metastases compared with regular cells at 21 days. TGF-β and HGF induced EMT expression, but signal transduction was quite different. TGF-β induced ERK, but not Akt phosphory-lation. HGF mediated both ERK and Akt phosphorylation. Akt inhibitor blocked Akt phosphorylation but did not affect TGF-β-induced activation of ERK, Snail and Slug. U-0126 did not reduce Snail activity by TGF-β at a concentration to block ERK phosphorylation. However, Akt inhibitor and U-0126 completely inhibited HGF-induced Slug activation. 5-FU mediated cell death in the EMT process induced by TGF-β more effectively than HGF. ERK/Akt signaling, but not the Smad pathway, may be one of the main processes in HGF-induced EMT, despite the Smad pathway, but not ERK/Akt, being critical for TGF-β-induced EMT. The MAPK/Akt pathway is indispensable in HGF/c-Met signaling. The ERK/Akt pathway particularly may be critical in the HGF-induced EMT process. However, long-term use of chemotherapeutic agents may induce drug resistance and distant metastases through EMT-related signaling pathway activation.

Clinical significance of glycoprotein nonmetastatic B and its association with HER2 in breast cancer

Glycoprotein nonmetastatic B (GPNMB) is a potential oncogene that is particularly expressed in melanoma and breast cancer (BC). To clarify its clinical significance in BC, we measured serum GPNMB in vivo and investigated its cross talk with human epidermal growth factor 2 (HER2). GPNMB was expressed in four of six breast cell lines (SK‐BR‐3, BT‐474, MDA‐MD‐231, and MDA‐MD‐157), two of six colorectal cell lines, and two of four gastric cancer (GC) cell lines. We established a GPNMB quantification system using enzyme‐linked immunosorbent assay (ELISA) for these cell lines. We measured serum GPNMB in vivo in 162 consecutive BC patients and in 88 controls (50 colorectal cancer [CC] and 38 GC patients). The GPNMB concentration in BC, CC and GC was 8.163, 5.751 and 6.55 ng/mL, respectively. The GPNMB level was significantly higher in BC patients than in CC patients (P = 0.021). The HER2‐rich subtype of BC patients had significantly higher GPNMB levels than other subtypes (vs. Luminal; P = 0.038; vs. DCIS; P = 0.0195). These high GPNMB levels decreased after treatment (surgery/chemotherapy). Next, we examined the relationship between GPNMB and HER2 in vitro using SK‐BR3 and BT‐474 (HER2‐positive/GPNMB‐positive) cells. GPNMB depletion by small interfering RNA (siRNA) increased both HER2 expression and phosphorylation. Trastuzumab (Tra) in combination with docetaxel promoted cell growth inhibition, and treatment with Tra or an Extracellular signal‐related kinase (ERK) inhibitor enhanced GPNMB expression. These results indicate that GPNMB might be a surrogate marker for BC and may cross talk with the HER2 signal pathway. GPNMB may therefore emerge as an important player in anti‐HER2 therapy.

Novel therapy for locally advanced triple-negative breast cancer

To evaluate a novel therapy for triple-negative breast cancer (TNBC), the biological responses to vitamin K3 (VK3) should be considered with the understanding of the features of breast cancer. In human breast cancer cell lines, the effects of VK3 on cell growth inhibition and the cellular signaling pathway were determined by MTT assay and western blotting. In the in vivo study, a subcutaneous tumor model of breast cancer was created, VK3 was injected into the subcutaneous tumors, and tumor size was measured. The IC50 of VK3 for breast cancer cells was calculated to be 11.3–25.1 μM. VK3 induced phosphorylation of whole tyrosine and epidermal growth factor receptor. VK3 mediated phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) for 30 min. ERK but not JNK phosphorylation was maintained for at least 6 h. In contrast, another antioxidant agent, catalase, showed no effect on either ERK phosphorylation or growth inhibition. On built-up tumors under the skin of mice, local treatment with VK3 was effective in a time- and dose-dependent manner, and the experiments for total tumor volume also showed a dose-dependent effect of VK3. The expression of phosphorylated ERK was clearly detected at 10.9 times the control in tumor tissue, whereas ethanol itself showed no effect. In conclusion, ERK plays a critical role in VK3-induced growth inhibition, and it will be the focus of next steps in the development of molecular therapy for TNBC.

Abstract 4319: Clinical significance and possible role of GPNMB in patients with breast cancer

Glycoprotein non-metastatic B (GPNMB) is a type I transmembrane protein, which is isolated from differential expression assay using metastatic melanoma cells. The physiological function is very little known but may be supposed to be associated with cell invasion and motility particularly in breast cancer cells. Here we investigated the role of GPNMB in breast cancer. First we checked expression of GPNMB by RT-PCR and western blot in several cancer cell lines including breast, gastric, and colon cancer followed by establishment of GPNMB measurement by ELISA because it9s reported that GPNMB is shed at extracellular domain by sheddase such as ADAM10. GPMNB expressed in breast (5/6:83%), gastric (3/6:50%), colon (1/7:14.3%) cancer cell lines. Of breast cancer cell lines, GPNMB was highly expressed in SK-BR3 (HER2 positive), BT474 (HER2/ER positive), MDA-MB-157 (Triple negative) cells. Shed GPNMB in culture medium was measurable and correlated with expression of each cell line. Next we evaluated serum GPNMB in patients with breast (n = 164; primary 119, metastatic 43), gastric (n = 38), and colorectal (n = 50) cancer in our institute from 2011.9-2014.2.) This study was approved by the central ethics committee of Gifu University. Serum GPNMBs were 9.403, 5.751, 6.550 ng/ml, respectively. GPNMB for breast cancer patients was statistically higher than those by colorectal cancer patients (p = 0.018). Of breast cancer patients, GPNMB for HER2-type patients was higher than those for Luminal type and DCIS patients (p = 0.0386, p = 0.0195, respectively). Those for triple negative patients was also higher than those for DCIS patients (p = 0.0459). Interestingly, serum GPNMB was dramatically reduced in accordance with chemotherapy in some patients. Based on these clinical observations, we further investigated relationship between GPNMP and HER2 in vitro. Blockage of GPNMB induced not only HER2 but also EGFR expression. On the other hand, inhibition of HER2 by trastuzumab increased expression of GPNMB. Depletion of GPNMB increased sensitivity of trastuzumab, suggesting that GPNMB may play an important role in crosstalk of signal transduction for breast cancer. These notions may suggest a novel therapeutic strategy to overcome HER2 positive breast cancer. Citation Format: Manabu Futamura, Masako Kanematsu, Atsuko Yamada, Kasumi Morimitsu, Akemi Morikawa, Ryutaro Mori, Kazuhiro Yoshida. Clinical significance and possible role of GPNMB in patients with breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4319. doi:10.1158/1538-7445.AM2015-4319