Ryutaro Mori

Contribution of thymidylate synthase to gemcitabine therapy for advanced pancreatic cancer.

Objectives: Thymidylate synthase (TS) inhibitors activate human equilibrative nucleoside transporter 1. We evaluated the contribution of TS expression to determine a treatment method providing an effect from gemcitabine (GEM). Methods: The expression of 5-fluorouracil (5-FU) and GEM metabolic factors (5-FU: TS, dihydropyrimidine dehydrogenase, orotate phosphoribosyltransferase; GEM: human equilibrative nucleoside transporter 1, deoxycytidine kinase, cytidine deaminase, 5&vprime;-nucleotidase) were studied in 7 pancreatic cancer cell lines by Western blotting, and drug resistance was evaluated by 3-[4,5-dimethylthiazol]-2,5-dephenyl tetrazolium bromide assay. The expression of 5-FU factors was observed immunohistochemically in resected pancreatic cancer specimens. Results: Gemcitabine concentrations that inhibited colony formation by 50% correlated with TS protein expression (P = 0.0169). With a 5-FU non-growth-inhibiting dose, GEM concentrations that inhibited colony formation by 50% were significantly reduced by one fourth to one tenth. Knockout of TS expression by small interfering RNA decreased resistance to GEM in the cell lines (P = 0.0019). Immunohistochemically, TS expression related to disease-free survival time of patients treated with GEM (P = 0.0224). A high expression of 5-FU factors was detected: orotate phosphoribosyltransferase: differentiated cases (P = 0.0137), lower T factor (P = 0.0411); dihydropyrimidine dehydrogenase: nerve invasion (P = 0.0188), lymph node recurrence (P = 0.0253); TS, positive N factor (P = 0.0061). Conclusions: The expression of TS provides an alternative source of substrate for DNA synthesis and positively correlates with GEM resistance and shortened patient survival. Abbreviations: GEM - gemcitabine, 5-FU - 5-fluorouracil, TS - thymidylate synthase, DPD - dihydropyrimidine dehydrogenase, OPRT - orotate phosphoribosyltransferase, hENT1 - human equilibrative nucleoside transporter 1

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.

Decreased FANCJ caused by 5FU contributes to the increased sensitivity to oxaliplatin in gastric cancer cells

BackgroundOxaliplatin is effective against many types of cancer, and the combination of 5-fluorouracil (5FU) and oxaliplatin is synergistically effective against gastric cancer, as well as colon cancer. The FANCJ protein is one of the Fanconi anemia (FA) gene products, and its interaction with the tumor suppressor BRCA1 is required for DNA double-strand break (DSB) repair. FANCJ also functions in interstrand crosslinks (ICLs) repair by linking to mismatch repair protein complex MLH1-PMS2 (MutLα). While oxaliplatin causes ICLs, 5FU is considered to cause DSBs. Therefore, we investigated the importance of FANCJ in the synergistic effects of oxaliplatin and 5FU in MKN45 gastric cancer cells and the derived 5FU-resistant cell line, MKN45/F2R.MethodsMKN1, TMK1, MKN45, and MKN45/F2R (5FU-resistant) gastric cancer cells were treated with 5FU and/or oxaliplatin. The signaling pathway was evaluated by a western blotting analysis and reverse transcription polymerase chain reaction (RT-PCR). Drug resistance was evaluated by the 3-(4,5-dimethyl-2-tetrazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay.ResultsIn MKN45 cells, the combination of 5FU and oxaliplatin had synergistic effects. DSBs appeared when the cells were treated with 5FU. FANCJ was down-regulated, and BRCA1 was induced in a dose- and time-dependent manner. MKN45 cells showed increased sensitivity to oxaliplatin when FANCJ was knocked down by short interfering (si) RNA. However, these findings were not observed in MKN45/F2R 5FU-resistant cells.ConclusionThese results strongly suggest that the decrease in FANCJ caused by 5FU treatment leads to an increase in sensitivity to oxaliplatin, thus indicating that the FANCJ protein plays an important role in the synergism of the combination of 5FU and oxaliplatin.

Prediction of macrometastasis in axillary lymph nodes of patients with invasive breast cancer and the utility of the SUV lymph node/tumor ratio using FDG-PET/CT

BackgroundAxillary lymph node dissection (ALND) is important for improving the prognosis of patients with node-positive breast cancer. However, ALND can be avoided in select micrometastatic cases, preventing complications such as lymphedema or paresthesia of the upper limb. To appropriately omit ALND from treatment, evaluation of the axillary tumor burden is critical. The present study evaluated a method for preoperative quantification of axillary lymph node metastasis using positron emission tomography/computed tomography (PET/CT).MethodsThe records of breast cancer patients who received radical surgery at the Gifu University Hospital (Gifu, Japan) between 2009 and 2014 were reviewed. The axillary lymph nodes were preoperatively evaluated by PET/CT. Lymph nodes were dissected by sentinel lymph node biopsy (SLNB) or ALND and were histologically diagnosed by experienced pathologists. The maximum standardized uptake value (SUVmax) was measured in both the axillary lymph node (SUV-LN) and primary tumor (SUV-T). The SUV-LN/T ratio (NT ratio) was calculated by dividing the SUV-LN by the SUV-T, and the efficacies of the NT ratio and SUV-LN were compared using receiver operating characteristic (ROC) curve analysis. The diagnostic performance was also compared between the techniques with the McNemar test.ResultsA total of 171 operable invasive breast cancer patients were enrolled, comprising 69 node-positive patients (macrometastasis (Mac): n = 55; micrometastasis (Mic): n = 14) and 102 node-negative patients (Neg). The NT ratio for node-positive patients was significantly higher than in node-negative patients (0.5 vs. 0.316, respectively, P = 0.041). The NT ratio for Mac patients (0.571) was significantly higher than in Mic (0.227) and Neg (0.316) patients (P <0.01 and P = 0.021, respectively). The areas under the curves (AUCs) by ROC analysis for the NT ratio and SUV-LN were 0.647 and 0.811, respectively (P <0.01). In patients with an SUV-T ≥2.5, the modified AUCs for the NT ratio and SUV-LV were 0.757 and 0.797 (not significant).ConclusionThe NT ratio and SUV-LN are significantly higher in patients with axillary macrometastasis than in those with micrometastasis or no metastasis. The NT ratio and SUV-LN can help quantify axillary lymph node metastasis and may assist in macrometastasis identification, particularly in patients with an SUV-T ≥2.5.

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.

Combination therapy with zoledronic acid and cetuximab effectively suppresses growth of colorectal cancer cells regardless of KRAS status

Targeted molecular therapy is an effective anticancer strategy. Anti‐EGFR monoclonal antibodies such as cetuximab (CTX) have been approved for the treatment of various malignancies, including colorectal cancer (CRC) with wild‐type KRAS. However, their efficacy in patients with KRAS mutations has not been established. Therefore, we investigated whether CTX treatment was effective as a single agent or in combination with zoledronic acid (ZOL) in human CRC cell lines with different KRAS status. CRC cell lines SW48 (wild‐type KRAS) and LS174T (mutant KRAS) were treated with ZOL, CTX and a combination of both drugs. Cytotoxicity was measured using the MTT assay. Changes in the levels of intracellular signaling proteins were evaluated using western blot analysis. Finally, we evaluated the efficacy of the combination treatment in an in vivo xenograft model. We observed that ZOL apparently inhibited growth in both cell lines, whereas CTX showed little effect. ZOL also increased the levels of unprenylated RAS. Combined ZOL and CTX treatment was synergistic in both cell lines and was associated with inhibition of the RAS‐MAPK and AKT‐mTOR signaling pathways. Furthermore, the combination treatment was more effective in suppressing the growth of xenografts derived from both SW48 and LS174T cells; this effect was associated with increased apoptosis. These results demonstrate that ZOL inhibits the growth of colon cancer cells regardless of KRAS status, and combination therapy using ZOL and CTX enhances this growth suppression. These findings suggest a novel strategy for the treatment of CRC independent of KRAS mutational status.

5FU resistance caused by reduced fluoro‑deoxyuridine monophosphate and its reversal using deoxyuridine

The mechanism of 5-fluorouracil (5FU) resistance was investigated, focusing on the level of thymidylate synthase (TS) ternary complex formed with fluoro-deoxyuridine monophosphate (FdUMP). MKN45 and 5FU-resistant MKN45/F2R cells were treated with 5FU and fluoro-deoxyuridine (FdU) in combination with deoxyuridine (dU) and thymidine (dT). Subsequently, the levels of ternary complex were determined by western blotting and the cell viability was calculated using an MTT assay. MKN45/F2R cells exhibited 5FU resistance (56.2-fold relative to MKN45 cells), and demonstrated decreased orotate phosphoribosyltransferase (OPRT) and increased TS levels, requiring a higher concentration of 5FU to induce ternary complex formation than MKN45 cells. Following transfection of small interfering RNA against OPRT, MKN45 exhibited increased resistance to 5FU and decreased ternary complex formation subsequent to treatment with 5FU, indicating that decreased OPRT led to increased 5FU resistance. However, MKN45/F2R also exhibited resistance to FdU, which can be converted to FdUMP without OPRT, and there was decreased ternary complex formation after treatment with FdU, indicating that the 5FU-resistant cells had the ability to decrease intracellular FdUMP. The addition of dU and thymidine dT to 5FU promoted the formation of ternary complexes and reversed 5FU resistance in MKN45/F2R cells, although dT inhibited the efficacy of raltitrexed (another TS inhibitor). These results suggested that 5FU-resistant cells had the ability to reduce intracellular FdUMP irrespective of decreased OPRT, which led to resistance to 5FU. This resistance was then inhibited by treatment with dT or dU.

Possible role of p53/Mieap-regulated mitochondrial quality control as a tumor suppressor in human breast cancer

Mitochondria‐eating protein (Mieap), encoded by a p53‐target gene, plays an important role in mitochondrial quality control (MQC). Mieap has been reported to have a critical role in tumor suppression in colorectal cancer. Here, we investigated its role as a tumor suppressor in breast cancer. The enforced expression of exogenous Mieap in breast cancer cells induced caspase‐dependent apoptosis, with activation of both caspase‐3/7 and caspase‐9. Immunohistochemistry revealed endogenous Mieap in the cytoplasm in 24/75 (32%) invasive ductal carcinomas (IDC), 15/27 (55.6%) cases of ductal carcinoma in situ (DCIS) and 16/18 (88.9%) fibroadenomas (FA) (IDC vs DCIS; P = 0.0389, DCIS vs FA; P = 0.0234, IDC vs FA; P < 0.0001). In IDC, the Mieap promoter was methylated in 6/46 (13%) cases, whereas p53 was mutated in 6/46 (13%) cases. Therefore, the p53/Mieap‐regulated MQC pathway was inactivated in 12/46 IDC (26.1%). Interestingly, all tumors derived from the 12 patients with Mieap promoter methylation or p53 mutations pathologically exhibited more aggressive and malignant breast cancer phenotypes. Impairment of p53/Mieap‐regulated MQC pathway resulted in significantly shorter disease‐free survival (DFS) (P = 0.021), although p53 status is more prognostic in DFS than Mieap promoter methylation. These results indicate that p53/Mieap‐regulated MQC has a critical role in tumor suppression in breast cancer, possibly in part through mitochondrial apoptotic pathway.

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

The efficacy of second-line hormone therapy for recurrence during adjuvant hormone therapy for breast cancer

Objectives: The recurrence of breast cancer during adjuvant hormone therapy is often targeted by second-line hormone therapy. However, there has been a lack of prior success with such treatments. We retrospectively investigated the efficacy of subsequent hormone therapy. Methods: Patients who underwent breast cancer surgery between 2006 and 2012 at our institution were investigated. Results: A total of 20 patients developed recurrence during adjuvant hormone therapy. There were four patients with luminal A, seven with luminal B and six with luminal HER2 tumors, respectively. Twelve patients received subsequent hormone therapy, and eight patients received chemotherapy. Subsequent hormone therapy produced one partial response (PR), two long stable disease (SD), one SD and five progressive disease (PD). A clinical benefit (CB) was obtained by 33%. Subsequent chemotherapy produced one complete response (CR), two PRs, one long SD and two PD, resulting in a CB in 66%. Among those who received any hormone therapy, the best responses were two PR, three long SD and one SD. A CB was obtained by 38%, while seven patients did not have any CB from hormone therapy. Meanwhile, the best responses to chemotherapy were two CRs, four PRs, three SD and two PD, thus resulting in a CB in 72%. All luminal A cases obtained a long SD or SD with hormone therapy. However, the CB of hormone therapy for non-luminal A cases was only 30%. Conclusions: The efficacy of hormone therapy for recurrence during adjuvant hormone therapy is poor, and when selecting therapy for such patients, the breast cancer subtype should be taken into account.