Arata Shimo

Involvement of kinesin family member 2C/mitotic centromere‐associated kinesin overexpression in mammary carcinogenesis

To elucidate the molecular mechanisms of mammary carcinogenesis and discover novel therapeutic targets for breast cancer, we previously carried out genome‐wide expression profile analysis of 81 breast cancer cases by means of cDNA microarray coupled with laser microbeam microdissection of cancer cells. Among the dozens of transactivated genes, in the present study we focused on the functional significance of kinesin family member 2C (KIF2C)/mitotic centromere‐associated kinesin (MCAK) in the growth of breast cancer cells. Northern blot and immunohistochemical analyses confirmed KIF2C/MCAK overexpression in breast cancer cells, and showed that it is expressed at undetectable levels in normal human tissues except the testis, suggesting KIF2C/MCAK to be a cancer–testis antigen. Western blot analysis using breast cancer cell lines revealed a significant increase in the endogenous KIF2C/MCAK protein level and its phosphorylation in G2/M phase. Treatment of breast cancer cells with small interfering RNA against KIF2C/MCAK effectively suppressed KIF2C/MCAK expression and inhibited the growth of the breast cancer cell lines T47D and HBC5. In addition, we found that KIF2C/MCAK expression was significantly suppressed by ectopic introduction of p53. These findings suggest that overexpression of KIF2C/MCAK might be involved in breast carcinogenesis and is a promising therapeutic target for breast cancers. (Cancer Sci 2008; 99: 62–70)

Oncogenic Role of MPHOSPH1, a Cancer-Testis Antigen Specific to Human Bladder Cancer

To disclose the molecular mechanism of bladder cancer, the second most common genitourinary tumor, we had previously done genome-wide expression profile analysis of 26 bladder cancers by means of cDNA microarray representing 27,648 genes. Among genes that were significantly up-regulated in the majority of bladder cancers, we here report identification of M-phase phosphoprotein 1 (MPHOSPH1) as a candidate molecule for drug development for bladder cancer. Northern blot analyses using mRNAs of normal human organs and cancer cell lines indicated this molecule to be a novel cancer-testis antigen. Introduction of MPHOSPH1 into NIH3T3 cells significantly enhanced cell growth at in vitro and in vivo conditions. We subsequently found an interaction between MPHOSPH1 and protein regulator of cytokinesis 1 (PRC1), which was also up-regulated in bladder cancer cells. Immunocytochemical analysis revealed colocalization of endogenous MPHOSPH1 and PRC1 proteins in bladder cancer cells. Interestingly, knockdown of either MPHOSPH1 or PRC1 expression with specific small interfering RNAs caused a significant increase of multinuclear cells and subsequent cell death of bladder cancer cells. Our results imply that the MPHOSPH1/PRC1 complex is likely to play a crucial role in bladder carcinogenesis and that inhibition of the MPHOSPH1/PRC1 expression or their interaction should be novel therapeutic targets for bladder cancers. [Cancer Res 2007;67(7):3276–85]

Elevated expression of protein regulator of cytokinesis 1, involved in the growth of breast cancer cells

To elucidate molecular mechanisms of mammary carcinogenesis and discover novel therapeutic targets for breast cancer, we previously carried out a genome‐wide expression profile analysis of 81 breast cancer cases by means of a combination of cDNA microarray and laser microbeam microdissection. Among the upregulated genes, we focused on the functional significance of protein regulator of cytokinesis 1 (PRC1) in the development of breast cancer. Western blot analysis using breast cancer cell lines revealed a significant increase in endogenous PRC1 levels in G2/M phase. Treatment of breast cancer cells with small interfering RNA against PRC1 effectively suppressed its expression and inhibited the growth of breast cancer cell lines T47D and HBC5. Furthermore, we found an interaction between PRC1 and kinesin family member 2C/mitotic centromere‐associated kinesin (KIF2C/MCAK) by coimmunoprecipitation and immunoblotting using COS‐7 cells, in which these molecules were introduced exogenously. These findings suggest the involvement of a PRC1–KIF2C/MCAK complex in breast tumorigenesis, and this complex should be a promising target for the development of novel treatments for breast cancer. (Cancer Sci 2007; 98: 174–181)

Involvement of elevated expression of multiple cell-cycle regulator, DTL/RAMP (denticleless/RA-regulated nuclear matrix associated protein), in the growth of breast cancer cells.

To investigate the detailed molecular mechanism of mammary carcinogenesis and discover novel therapeutic targets, we previously analysed gene expression profiles of breast cancers. We here report characterization of a significant role of DTL/RAMP (denticleless/RA-regulated nuclear matrix associated protein) in mammary carcinogenesis. Semiquantitative RT–PCR and northern blot analyses confirmed upregulation of DTL/RAMP in the majority of breast cancer cases and all of breast cancer cell lines examined. Immunocytochemical and western blot analyses using anti-DTL/RAMP polyclonal antibody revealed cell-cycle-dependent localization of endogenous DTL/RAMP protein in breast cancer cells; nuclear localization was observed in cells at interphase and the protein was concentrated at the contractile ring in cytokinesis process. The expression level of DTL/RAMP protein became highest at G1/S phases, whereas its phosphorylation level was enhanced during mitotic phase. Treatment of breast cancer cells, T47D and HBC4, with small-interfering RNAs against DTL/RAMP effectively suppressed its expression and caused accumulation of G2/M cells, resulting in growth inhibition of cancer cells. We further demonstrate the in vitro phosphorylation of DTL/RAMP through an interaction with the mitotic kinase, Aurora kinase-B (AURKB). Interestingly, depletion of AURKB expression with siRNA in breast cancer cells reduced the phosphorylation of DTL/RAMP and decreased the stability of DTL/RAMP protein. These findings imply important roles of DTL/RAMP in growth of breast cancer cells and suggest that DTL/RAMP might be a promising molecular target for treatment of breast cancer.

Activation of an estrogen/estrogen receptor signaling by BIG3 through its inhibitory effect on nuclear transport of PHB2/REA in breast cancer

Breast cancer is known to be a hormone‐dependent disease, and estrogens through an interaction with estrogen receptor (ER) enhance the proliferative and metastatic activity of breast tumor cells. Here we show a critical role of transactivation of BIG3, brefeldin A‐inhibited guanine nucleotide‐exchange protein 3, in activation of the estrogen/ER signaling in breast cancer cells. Knocking‐down of BIG3 expression with small‐interfering RNA (siRNA) drastically suppressed the growth of breast cancer cells. Subsequent coimmunoprecipitation and immunoblotting assays revealed an interaction of BIG3 with prohibitin 2/repressor of estrogen receptor activity (PHB2/REA). When BIG3 was absent, stimulation of estradiol caused the translocation of PHB2/REA to the nucleus, enhanced the interaction of PHB2/REA and ERα, and resulted in suppression of the ERα transcriptional activity. On the other hand, when BIG3 was present, BIG3 trapped PHB2/REA in the cytoplasm and inhibited its nuclear translocation, and caused enhancement of ERα transcriptional activity. Our results imply that BIG3 overexpression is one of the important mechanisms causing the activation of the estrogen/ERα signaling pathway in the hormone‐related growth of breast cancer cells. (Cancer Sci 2009)

Involvement of G-patch domain containing 2 overexpression in breast carcinogenesis

Through analysis of the detailed genome‐wide gene expression profiles of 81 breast tumors, we identified a novel gene, G‐patch domain containing 2 (GPATCH2), that was overexpressed in the great majority of breast cancer cases. Treatment of breast cancer cells MCF‐7 and T47D with siRNA against GPATCH2 effectively suppressed its expression, and resulted in the growth suppression of cancer cells, suggesting its essential role in breast cancer cell growth. We found an interaction of GPATCH2 protein with hPrp43, an RNA‐dependent ATPase. Their interaction could significantly enhance the ATPase activity of hPrp43 and induce a growth‐promoting effect on mammalian cells. Because northern blot analyses of normal human organs implied GPATCH2 to be a novel cancer/testis antigen, targeting GPATCH2 or inhibition of the interaction between GPATCH2 and hPrp43 could be a promising novel therapeutic strategy of breast cancer. (Cancer Sci 2009)

Comparison between Ki67 labeling index determined using image analysis software with virtual slide system and that determined visually in breast cancer

AbstractBackgroundnIn recent papers, Ki67 labeling index (LI) has been used to classify breast cancer patients into the low and high Ki67LI groups for comparison studies, which showed significant differences in many prognostic factors. It has not been clarified whether image analysis software can be used for calculating LI in breast cancer. In our study, we examined whether Ki67LI in breast cancer calculated using image analysis software correlates with that measured on the basis of visual.MethodsFifty patients were randomly selected among breast cancer patients who underwent surgical operation from March, 2010 to May, 2010 in our hospital without preoperative chemotherapy. In this study, for the virtual slide system (VSS: VS120-L100, Olympus, Tokyo, Japan), the high-resolution VSs of all the 50 patients were prepared as samples. The image analysis software use for calculating LI was Tissuemorph Digital Pathology (Tissuemorph DP: Visiopharm, Hoersholm, Denmark). The calculated LI was extracted from 3 to 5 views containing hot spots. The LI calculated using Tissuemorph DP was designed as LI/image/T. The digital image of 3 to 5 LI/image/T views was printed out, and on the digital photograph, we counted visually the number of Ki67-immunopositive cells in exactly the same area, and the percentage of Ki67-immunopositive cells was designed as LI/direct. Moreover, a pathologist’s assistant (PA) determined the tumor area in the same specimen using VSS and calculated LI using Tissuemorph DP, which was designed as LI/image/PA. The chief pathologist (CP) similarly calculated LI which was designed as LI/image/CP. We evaluated the degree of agreement between different data sets “LI/image/T and LI/direct” and “LI/image/T, LI/image/CP, and LI/image/PA” by using interclass correlation coefficient (ICC).ResultsThe average counts of cells were as follows: LI/direct, 3209.7xa0±xa01970.4 (SD); LI/image/T, 2601.6xa0±xa01697.1; LI/image/PA, 2886.5xa0±xa02027.5; LI/image/CP, 18805.5xa0±xa022293.4. The values of LI/direct and LI/image/T showed almost perfect agreement as showed by an ICC of 0.885 (95xa0% CI, 0.806–0.933; pxa0<xa00.001). The agreement among three investigators was almost perfect. The obtained ICC was 0.825 (95xa0% CI, 0.739–0.890; pxa0<xa00.001) among the data of LI/image/T, LI/image/CP and LI/image/PA. There were five cases that immunopositivity for Ki67 showed a more than 10xa0% disagreement between LI/direct and LI/image/T.ConclusionThe merits of calculating Ki67 LI using Tissuemorph DP are as follows. First, the staining intensity of the cells to be counted can be adjusted. Second, the portion of a tumor including “hot spots” for counting can be chosen. Third, many cancer cells can be counted more rapidly using Tissuemorph DP than by visual observation. However, it is important that pathologist should check and carry out the final decision of the data, when Ki67 LI using Tissuemorph DP is calculated.

Oncologic outcomes and technical considerations of nipple-sparing mastectomies in breast cancer: experience of 425 cases from a single institution.

BackgroundNipple-sparing mastectomy (NSM) is an advantageous treatment option, providing a complete cure and good cosmetic results. We tested whether NSM is a surgically and oncologically safe technique.MethodsWe evaluated the oncological outcome of 425 breasts in 413 patients who underwent NSM between January 2000 and March 2013. We retrospectively reviewed patient data and analyzed all patient characteristics as potential risk factors of recurrence at the nipple–areola complex (NAC). To confirm the oncological safety of NSM, we compared outcomes of NSM and conventional total mastectomy.ResultsThe median follow-up time after surgery was 46.8xa0months (range 6–158xa0months). Nipple necrosis was observed in 6 cases (1.4xa0%). The cumulative local recurrence rate after NSM was 5.8xa0% (25/425 cases), similar to that of conventional total mastectomy in the same period (5.6xa0%, 49/878 cases). Furthermore, the cumulative local recurrence rate at the NAC was 2.3xa0% (10 cases). HER2-enriched tumors and young age (<40xa0years) were significant risk factors for recurrence at the NAC. In patients with recurrence, the site of recurrence was easily excised, and good cosmetic results were achieved in breast reconstruction cases.ConclusionNSM is safe with a low complication rate. No significant difference was observed in cumulative local recurrence rate, cumulative distant disease recurrence rate, and overall survival between patients who underwent NSM or conventional total mastectomy, confirming that NSM was surgically and oncologically safe.

Significant Effect of Polymorphisms in CYP2D6 on Response to Tamoxifen Therapy for Breast Cancer; a Prospective Multicenter Study.

Purpose: CYP2D6 is the key enzyme responsible for the generation of the potent active metabolite of tamoxifen, “endoxifen.” There are still controversial reports questioning the association between CYP2D6 genotype and tamoxifen efficacy. Hence, we performed a prospective multicenter study to evaluate the clinical effect of CYP2D6 genotype on tamoxifen therapy. Experimental Design: We enrolled 279 patients with hormone receptor–positive and human epidermal growth factor receptor 2-negative, invasive breast cancer receiving preoperative tamoxifen monotherapy for 14 to 28 days. Ki-67 response in breast cancer tissues after tamoxifen therapy was used as a surrogate marker for response to tamoxifen. We prospectively investigated the effects of allelic variants of CYP2D6 on Ki-67 response, pathological response, and hot flushes. Results: Ki-67 labeling index in breast cancer tissues significantly decreased after preoperative tamoxifen monotherapy (P = 0.0000000000000013). Moreover, proportion and Allred scores of estrogen receptor–positive cells in breast cancer tissues were significantly associated with Ki-67 response (P = 0.0076 and 0.0023, respectively). Although CYP2D6 variants were not associated with pathologic response nor hot flushes, they showed significant association with Ki-67 response after preoperative tamoxifen therapy (P = 0.018; between two groups, one with at least one wild-type allele and the other without a wild-type allele). Conclusions: This is the first prospective study evaluating the relationship between CYP2D6 variants and Ki-67 response after tamoxifen therapy. Our results suggest that genetic variation in CYP2D6 is a key predictor for the response to tamoxifen in patients with breast cancer. Clin Cancer Res; 23(8); 2019–26. ©2016 AACR.

Positive predictive value for malignancy of pure flat epithelial atypia diagnosis by percutaneous needle biopsy of the breast: management of FEA in ultrasonography

BackgroundSome reports suggest that the rate of definitive diagnosis of malignant tumors, namely, the final diagnosis being revised to a higher stage, in patients diagnosed as having flat epithelial atypia (FEA) by percutaneous needle biopsy of the breast (PNB) is as low as 0–3xa0%. However, other reports suggest that the rate is as high as 10xa0% or more, bringing confusion on this issue. We examined the positive predictive value for malignancy in the patients diagnosed as having pure FEA and the patients’ radiolopathological characteristics observed in our hospital.MethodsOf the patients who underwent PNB in our facility, those who were diagnosed as having pure FEA were recruited as the subjects of this study.ResultsOf the 4,197 consecutive patients who underwent PNB, 44 (1.0xa0%) were diagnosed as having pure FEA following a re-examination. Among 44 cases, 39 cases were selected as the subjects of this study. Among the 39 patients, six patients were diagnosed as having malignant lesions, two of whom had invasive carcinoma of no special type (papillotubular type), one had tubular carcinoma, one had ductal carcinoma in situ (DCIS) of high nuclear grade, one had DCIS of intermediate nuclear grade, and one had DCIS of low nuclear grade. The diameters of 6 malignant lesions were 10–30xa0mm at ultrasonography (US) examination. Five of the 39 patients had contralateral breast cancer.ConclusionThe positive predictive value for malignancy of pure FEA was 15.7xa0%. The patients with pure FEA may make a follow up without an excisional biopsy when the lesion sizes less than 10xa0mm on US examination.