Masahiro Kashiwaba

Hypoxia-induced Jagged2 promotes breast cancer metastasis and self-renewal of cancer stem-like cells.

Notch signaling is often and aberrantly activated by hypoxia during tumor progression; however, the exact pathological role of hypoxia-induced Notch signaling in tumor metastasis is as yet poorly understood. In this study, we aimed to define the mechanism of Notch-ligand activation by hypoxia in both primary tumor and bone stromal cells in the metastatic niche and to clarify their roles in tumor progression. We have analyzed the expression profiles of various Notch ligands in 779 breast cancer patients in GEO database and found that the expression of Jagged2 among all five ligands is most significantly correlated with the overall- and metastasis-free survival of breast cancer patients. The results of our immunohistochemical (IHC) analysis for Jagged2 in 61 clinical samples also revealed that both Jagged2 and Notch signaling were strongly upregulated at the hypoxic invasive front. Activation of Jagged2 by hypoxia in tumor cells induced EMT and also promoted cell survival in vitro. Notably, a γ-secretase inhibitor significantly blocked Notch-mediated invasion and survival under hypoxia by promoting expression of E-cadherin and inhibiting Akt phosphorylation. Importantly, Jagged2 was also found to be upregulated in bone marrow stroma under hypoxia and promoted the growth of cancer stem-like cells by activating their Notch signaling. Therefore, hypoxia-induced Jagged2 activation in both tumor invasive front and normal bone stroma has a critical role in tumor progression and metastasis, and Jagged2 is considered to be a valuable prognostic marker and may serve as a novel therapeutic target for metastatic breast cancer.

Resveratrol suppresses growth of cancer stem-like cells by inhibiting fatty acid synthase.

Resveratrol is a natural polyphenolic compound and has been shown to exhibit cardio-protective as well as anti-neoplastic effects on various types of cancers. However, the exact mechanism of its anti-tumor effect is not clearly defined. Resveratrol has been shown to have strong hypolipidemic effect on normal adipocytes and as hyper-lipogenesis is a hallmark of cancer cell physiology, the effect of resveratrol on lipid synthesis in cancer stem-like cells (CD24−/CD44+/ESA+) that were isolated from both ER+ and ER− breast cancer cell lines was examined. The authors found that resveratrol significantly reduced the cell viability and mammosphere formation followed by inducing apoptosis in cancer stem-like cells. This inhibitory effect of resveratrol is accompanied by a significant reduction in lipid synthesis which is caused by the down-regulation of the fatty acid synthase (FAS) gene followed by up-regulation of pro-apoptotic genes, DAPK2 and BNIP3. The activation of apoptotic pathway in the cancer stem-like cells was suppressed by TOFA and by Fumonisin B1, suggesting that resveratrol-induced apoptosis is indeed through the modulation of FAS-mediated cell survival signaling. Importantly, resveratrol was able to significantly suppress the growth of cancer stem-like cells in an animal model of xenograft without showing apparental toxicity. Taken together, the results of this study indicate that resveratrol is capable of inducing apoptosis in the cancer stem-like cells through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol.

Clinical Significance of the 21-Gene Signature (Oncotype DX) in Hormone Receptor-Positive Early Stage Primary Breast Cancer in the Japanese Population

The 21‐gene signature has been intensively studied and incorporated into major guidelines for treatment decision in early breast caner. However, it remains to be examined whether this system is applicable to Asian populations.

Aberrations of theAPC gene in primary breast carcinoma

Aberrations of theAPC gene, which plays an important role in the genesis of familial adenomatous polyposis and colorectal carcinoma, were investigated in 31 surgical specimens of primary breast carcinoma. These studies utilized the polymerase chain reaction followed by restriction-fragment-length polymorphism and single-strand-conformation polymorphism analyses combined with tumor cell enrichment by cell sorting. Loss of heterozygosity at theAPC locus was detected in 8 (38%) of 21 informative cases, but only 2 (6%) of 31 tumors carried a mutatedAPC gene. Direct DNA sequencing analysis confirmed mutations at codon 1081 (AGC to ATC) resulting in an amino acid substitution of serine for isoleucine, and at codon 1096 (CAG to CAT) resulting in a substitution of glutamine for histidine. There were no significant correlations between the loss of heterozygosity or mutation at theAPC locus and any clinicopathological characteristics. Our present observations suggest that the mutations of theAPC gene may play an important role in the genesis of certain breast carcinomas, and that another tumor-suppressor gene, which is the true target of frequent loss of heterozygosity, may exist near theAPC gene.

Quantitative Measurement of Thymidylate Synthase and Dihydropyrimidine Dehydrogenase mRNA Level in Gastric Cancer by Real-time RT-PCR

We used real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) to assay expression of the mRNA of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in gastric cancer tissue with the objective of establishing a system to measure TS and DPD in ultra‐low‐volume samples. Nude mouse xenografts of 5 human gastric cancer cell lines and 85 clinical samples were used as the specimens in this study. Sensitivity to 5‐fluorouracil (5‐FU) was determined on the basis of the relative tumor proliferation rate in mice and the results of ATP assay using serum‐free cultures of the clinical samples. mRNA expression was measured in tumor tissue by real‐tune RT‐ PCR using the ABI PRISM 7700 system. The values for expression of the mRNA for TS and DPD were corrected according to the level of glyceraldehyde‐3‐phosphate dehydrogenase mRNA expression. The xenografts yielded correlations between TS and DPD mRNA expression and the activity of the enzymes (TS: rs=0.700, DPD: rs=0.900), and an inverse correlation was noted between the mRNA levels and sensitivity to 5‐FU (TS: rs=‐0.900, DPD: rs=‐0.800). The clinical samples showed an inverse correlation between 5‐FU sensitivity and mRNA expression (TS: rs=‐0.518, DPD: rs=‐0.564). Sensitivity to 5‐FU was noted only in cases in which TS mRNA expression and DPD mRNA expression were both low. Real‐time RT‐PCR can provide a highly sensitive assessment of TS and DPD mRNA expression in gastric cancer, and it was useful for predicting 5‐FU sensitivity.

Epithelial-cadherin gene is not mutated in ductal carcinomas of the breast.

We investigated mutations of the epithelial (E)‐cadherin gene and loss of heterozygosity (LOH) at flanking loci using three microsatellite markers on the long arm of chromosome 16 in 25 ductal carcinomas of the breast. Expression of E‐cadherin was also investigated immunohistochemically. No mutations were detected in exons 6 through 9 of the E‐cadherin gene. LOH was observed more frequently (42%) at D16S402 (16q‐ter) than at D16S421 (16q22.3‐q23.1) (17%), which is located near the E‐cadherin gene. Expression of E‐cadherin was observed at the cell borders in 92% (11/12) of the tumors examined. The absence of mutations in the E‐cadherin gene and its conserved expression suggest that inactivation of E‐cadherin does not contribute significantly to the invasion or metastatic potential of ductal carcinomas of the breast. Furthermore, the high frequency of LOH at 16q‐ter suggests the existence of another tumor suppressor gene which may play a crucial role in the genesis of ductal carcinomas of the breast.

Evaluation of Trastuzumab Without Chemotherapy as a Post-operative Adjuvant Therapy in HER2-positive Elderly Breast Cancer Patients: Randomized Controlled Trial [RESPECT (N-SAS BC07)]†

OBJECTIVE This trial is conducted to investigate the benefit of trastuzumab monotherapy compared with a combination therapy of trastuzumab and chemotherapy in women over 70 years with human epidermal growth factor receptor type-2-positive primary breast cancer. METHODS Inclusion criteria are the following: histologically diagnosed as invasive breast cancer and received curative operation for primary breast cancer; Stage I, IIA, IIB or IIIA/M0; and baseline left ventricular ejection fraction is ≥55%. Patients are randomized to receive either trastuzumab (8 mg/kg loading dose, 6 mg/kg every 3 weeks for 1 year) plus chemotherapy selected from regimens specified on the protocol or trastuzumab monotherapy. The primary endpoint is disease-free survival. Secondary endpoints are overall survival, relapse-free survival, safety, health-related quality of life, comprehensive geriatric assessment and cost effectiveness. RESULTS Patients recruitment has been commenced in October 2009. Enrollment of 300 patients is planned during the 4-year recruitment period. CONCLUSIONS We hereby report the study concept.

Improved detection of loss of heterozygosity at retinoblastoma gene locus in human breast carcinoma

Loss of heterozygosity (LOH) at the retinoblastoma (Rb) gene locus was investigated in 33 breast carcinomas by polymerase chain reaction single‐strand conformation polymorphism (PCR‐SSCP) analysis after tumor cell enrichment by cell sorting. The efficacy of cell sorting was evaluated by comparing the results of PCR‐SSCP with and without cell sorting. Ten of 17 (59%) informative cases showed LOH at this locus by cell sorting combined with PCR‐SSCP, although LOH was detectable in six (35%) cases without cell sorting. Flow cytometry and histologic examination revealed that this underestimation may occur when the tumor cell population is less than 50% in the specimens analyzed. It is concluded that LOH of the Rb gene occurs more frequently in human breast carcinoma than previously thought, and thus may contribute significantly to the development and/or progression of this tumor.

Frequent loss of heterozygosity at the deleted in colorectal carcinoma gene locus and its association with histologic phenotypes in breast carcinoma

Loss of heterozygosity (LOH) at the deleted in colorectal carcinoma gene (DCC), a tumour suppressor gene that encodes a protein with high homology to the neural cell adhesion molecule, was investigated in 42 surgical specimens of primary breast carcinoma. LOH was analysed in breast carcinoma by amplifying the DNA, spanning a variable number of tandem repeats site and a restriction fragment length polymorphism site within DCC, using the polymerase chain reaction (PCR). Cell sorting was used to enrich carcinoma cells. The expression of the DCC gene was also investigated using a reverse transcription-PCR method followed by Southern blot hybridization. LOH at the DCC locus was detected in 15 (51.7%) of 29 informative cases and 10 of 13 cases having DCC-LOH showed distinct reduction or loss of DCC expression. The DCC-LOH was closely associated with certain histological phenotypes; DCC-LOH was more frequent in scirrhous carcinomas than in solid-tubular ones (P<0.05), and was also more frequent in carcinomas with infiltration into fat tissue over the mammary gland than in those without infiltration (P<0.05). DCC-LOH was detected in invasive lobular carcinomas (2/2), but in none of the noninvasive ductal carcinomas (0/2). These observations suggest that malignant histological phenotypes are associated with DCC-LOH.

Pulmonary Hilar Lymph Node Metastasis of Breast Cancer Induced Bronchopleural Fistula and Superior Vena Cava Syndrome

Patient: Female, 56 Final Diagnosis: Broncho-pleural fistula • empyema • supra-vena cava syndrome Symptoms: Dyspnea • fever • facial edema Medication: — Clinical Procedure: — Specialty: Oncology and Pulmonology Objective: Unusual clinical course Background: It is extremely rare for pulmonary hilar lymph node metastasis (PHLNM) of a cancer to be independently lethal. Here, we report an exceedingly rare case of cavitation in PHLNM from breast cancer triggering bronchopleural fistula and empyema (BPFE), complicated with superior vena cava syndrome (SVCS). Case Report: A 56-year-old woman who had undergone left segmental mastectomy and axillary lymph node dissection due to left breast cancer was then treated for 1 year with postoperative adjuvant chemotherapy. Recurrence of right PHLNM was observed 2 years after the operation, for which 3 courses of bevacizumab (BEV) and paclitaxel combination chemotherapy were administered. The woman had dyspnea and fever during the washout period, and CT examination revealed fistula formation between the right PHLNM cavitation and right main bronchus, so she was admitted for further treatment. This fistula rapidly progressed to BPFE, and contralateral aspiration was observed to cause pneumonia of the left lung. In addition, edema of both upper limbs and head and neck were observed, and CT examination revealed SVCS caused by re-enlargement of PHLNM. Active treatment was performed, but the recommencement of chemotherapy was not possible, and she died on Day 150 of admission. Conclusions: We think that PHLNM deteriorated to central necrosis due to chemotherapy with BEV taking effect, leading to formation of BPFE. The case was also made more difficult due to the complication of SVCS caused by the re-enlarged PHLNM.