Naofumi Kagara

Zinc transporter LIVI controls epithelial-mesenchymal transition in zebrafish gastrula organizer

Vertebrate gastrulation is a critical step in the establishment of body plan. During gastrulation, epithelial-mesenchymal transition (EMT) occurs. EMT is one of the central events of embryonic development, organ and tissue regeneration, and cancer metastasis. Signal transducers and activators of transcription (STATs) mediate biological actions such as cell proliferation, differentiation and survival in response to cytokines and growth factors, in a variety of biological processes. STATs are also important in EMT during gastrulation, organogenesis, wound healing and cancer progression. We previously showed that STAT3 is activated in the organizer during zebrafish gastrulation and its activity is essential for gastrulation movements. The requirement for STAT3 is cell-autonomous for the anterior migration of gastrula organizer cells, and non-cell-autonomous for the convergence of neighbouring cells. The molecular mechanisms of STATs action in EMT, however, are unknown. Here we identify LIV1, a breast-cancer-associated zinc transporter protein, as a downstream target of STAT3 that is essential and sufficient for STAT3s cell-autonomous role in the EMT of zebrafish gastrula organizer cells. Furthermore, we demonstrate that LIV1 is essential for the nuclear localization of zinc-finger protein Snail, a master regulator of EMT. These results establish a molecular link between STAT3, LIV1 and Snail in EMT.

Zinc and its transporter ZIP10 are involved in invasive behavior of breast cancer cells

Zinc is an essential element, necessary for sustaining all life. Zinc deficiency causes taste impairments, immune deficiency, skin problems, and growth and mental retardation. Recent reports suggest that zinc is associated with an increased risk of cancer, although it is still unclear whether zinc or its transporters are involved in cancer progression. Here we show that zinc and its transporter ZIP10 are involved in the invasive behavior of breast cancer cells. The screening of clinical samples for ZIP10 mRNA expression suggested that ZIP10 was significantly associated with the metastasis of breast cancer to the lymph node. In addition, the expression of ZIP10 mRNA was higher in the invasive and metastatic breast cancer cell lines MDA‐MB‐231 and MDA‐MB‐435S than in less metastatic breast cancer cell lines, such as MCF7, T47D, ZR75‐1 and ZR75‐30. In in vitro cell migration assays, the depletion of zinc transporter ZIP10 and intracellular zinc inhibited the migratory activity of MDA‐MB‐231 and MDA‐MB‐435S cells. These results showed that zinc and ZIP10 play an essential role in the migratory activity of highly metastatic breast cancer cells, and suggest ZIP10 as a possible marker for the metastatic phenotype of breast cancer and a promising target of novel treatment strategies. (Cancer Sci 2007; 98: 692–697)

Mutational analysis of MED12 in fibroadenomas and phyllodes tumors of the breast by means of targeted next-generation sequencing

We aimed to analyze MED12 mutation in fibroadenomas (FAs) and phyllodes tumors (PTs) of the breast, which are closely related and consist of epithelial and stromal components. Targeted deep-sequencing using next-generation sequencing was performed in FAs (n = 58) and PTs (n = 27). The frequency of MED12 mutant tumors was significantly higher (P = 0.016) in PTs (74.1 %) than in FAs. (46.6 %). As for FAs, this frequency was significantly higher (P = 0.001) for intracanalicular type (69.0 %) than for other histological subtypes such as pericanalicular, organoid, and mastopathic types (24.1 %). Laser microdissection study revealed that stromal cells, but not epithelial cells, harbored MED12 mutations in both FAs and PTs. MED12 mutation is implicated in the pathogenesis of both FAs and PTs. The similarly high frequency of MED12 mutation in intracanalicular type FAs suggests that they are most closely related to PTs. It is thus speculated that FAs with MED12 mutation are more likely to progress to PTs.

Construction of novel immune-related signature for prediction of pathological complete response to neoadjuvant chemotherapy in human breast cancer

BACKGROUND The aim of this study was to construct a novel prediction model for the pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) using immune-related gene expression data. PATIENTS AND METHODS DNA microarray data were used to perform a gene expression analysis of tumor samples obtained before NAC from 117 primary breast cancer patients. The samples were randomly divided into the training (n = 58) and the internal validation (n = 59) sets that were used to construct the prediction model for pCR. The model was further validated using an external validation set consisting of 901 patients treated with NAC from six public datasets. RESULTS The training set was used to construct an immune-related 23-gene signature for NAC (IRSN-23) that is capable of classifying the patients as either genomically predicted responders (Gp-R) or non-responders (Gp-NR). IRSN-23 was first validated using an internal validation set, and the results showed that the pCR rate for Gp-R was significantly higher than that obtained for Gp-NR (38 versus 0%, P = 1.04E-04). The model was then tested using an external validation set, and this analysis showed that the pCR rate for Gp-R was also significantly higher (40 versus 11%, P = 4.98E-23). IRSN-23 predicted pCR regardless of the intrinsic subtypes (PAM50) and chemotherapeutic regimens, and a multivariate analysis showed that IRSN-23 was the most important predictor of pCR (odds ratio = 4.6; 95% confidence interval = 2.7-7.7; P = 8.25E-09). CONCLUSION The novel prediction model (IRSN-23) constructed with immune-related genes can predict pCR independently of the intrinsic subtypes and chemotherapeutic regimens.

BRCA1 promoter methylation of normal breast epithelial cells as a possible precursor for BRCA1-methylated breast cancer

The breast cancer susceptibility gene 1 (BRCA1) and glutathione S‐transferase P1 (GSTP1) promoters are reportedly often methylated in breast cancer tissues. Their methylation status in surrounding normal breast tissues has not been examined thoroughly although this may well be important for a better understanding of breast carcinogenesis. In this study, BRCA1 and GSTP1 promoter methylation was examined by methylation‐specific PCR assay. Patients with BRCA1‐methylated (n = 15) or BRCA1‐unmethylated (n = 15) tumors and those with GSTP1‐methylated (n = 9) or GSTP1‐unmethylated (n = 11) tumors were included in the present study. Methylation status of manually micro‐dissected normal epithelial cells from the formalin‐fixed paraffin‐embedded sections of normal breast tissues adjacent to and distant from the tumors was examined at multiple sites (n = 1–5). Of the 15 patients with BRCA1‐methylated tumors, 9 harbored BRCA1 promoter methylation in at least one site of the normal breast tissues. However, no BRCA1 promoter methylation was observed at any site of the normal tissues of the 15 patients with BRCA1‐unmethylated tumors. No GSTP1 promoter methylation was observed in the normal tissues regardless of the methylation status of the tumors. The presence of BRCA1 promoter methylation in the normal tissues was confirmed in the epithelial cells enriched with the magnetic‐activated cell sorting method. Our findings suggest that a small proportion of normal breast epithelial cells with BRCA1 promoter methylation can be precursor cells from which BRCA1‐methylated breast tumors may originate. This does not apply to GSTP1 promoter methylation.

Methylated DNA and high total DNA levels in the serum of patients with breast cancer following neoadjuvant chemotherapy are predictive of a poor prognosis

In a previous study, we established a one-step methylation-specific polymerase chain reaction (OS-MSP) assay for the detection of methylated DNA (met-DNA) and total DNA levels in serum. For the present study, this OS-MSP assay was used for patients with breast cancer treated with neoadjuvant chemotherapy (NAC) in order to investigate the prognostic significance of met-DNA and total DNA levels. Following treatment with NAC and prior to surgery, serum samples obtained from 120 patients with stage II/III breast cancer were subjected to the OS-MSP assay for analysis of the glutathione S-transferase pi 1, Ras association (RalGDS/AF-6) domain family member 1 and retinoic acid receptor β2 genes. The detection of methylation in a minimum of one of these genes indicated a positive outcome of the assay. The total DNA content of the serum was also determined. Of the 120 stage II/III patients, seven (6%) were positive for met-DNA in serum and showed a significantly worse overall survival (OS) time compared with patients negative for met-DNA (n=113) (5-year OS, 43 vs. 85%; P=0.002). The patients with high total DNA levels in serum (n=40) also showed a significantly worse OS compared with those with low total DNA levels (n=80) (65 vs. 91%; P<0.001). The presence of met-DNA and high total DNA levels in the serum were found to be significant prognostic factors that are independent of a pathological complete response by multivariate analysis. Following NAC, met-DNA and high total DNA levels in the serum detected with the OS-MSP assay constitute novel prognostic factors for patients with breast cancer; this may be clinically useful for the prognosis prediction for patients who do not achieve a pathological complete response following NAC.

Clinicopathological analysis of breast ductal carcinoma in situ with ALDH1-positive cancer stem cells.

Objective: The aim of this study was to elucidate the clinicopathological characteristics of breast ductal carcinomas in situ (DCIS) with aldehyde dehydrogenase 1 (ALDH1)-positive cancer stem cells (CSCs). Methods: DCIS (n = 194) were subjected to immunohistochemical staining and results were examined for associations with various clinicopathological parameters. The ALDEFLUOR assay for breast cancer cell lines was also performed to determine the proportion of CSCs according to intrinsic subtype. Results: DCIS with ALDH1-positive CSCs were significantly (p < 0.05) more likely to be estrogen receptor-α (ER)-negative, progesterone receptor (PgR)-negative, Ki67-positive and HER2-positive tumors. Luminal A subtype (8.6%) showed a significantly (p < 0.001) lower ALDH1 positivity than the other subtypes [luminal B (50.0%), luminal HER2 (36.8%), HER2 (35.3%) and triple-negative subtype (26.7%)]. Double immunostaining revealed that ALDH1-positive CSCs did not overlap with ER-, PgR- or Ki67-positive tumor cells but did overlap with HER2-positive tumor cells. The percentage of ALDEFLUOR-positive CSCs was lower in the luminal A cell lines (0.02% for T-47D and 0.4% for MCF7) than in the luminal HER2 (9.1% for BT-474 and 9.5% for MDA-MB-361), HER2 (13.9% for AU565 and 33.2% for SK-BR-3) and triple-negative cell lines (28.4% for MDA-MB-231 and 30.7% for MDA-MB-468). Conclusions: Our results suggest that most CSCs in DCIS are in the G0 phase (Ki67 negative) and do not express ER or PgR, but they do express HER2 in HER2-positive tumors.

One-step nucleic acid amplification assay for intraoperative prediction of non-sentinel lymph node metastasis in breast cancer patients with sentinel lymph node metastasis

The aim of the present study was to construct the intra-operative prediction model of non-sentinel lymph node (non-SLN) metastasis in breast cancer patients with SLN metastasis using one-step nucleic acid amplification (OSNA). Of 833 breast cancer patients (T1-T2, N0) who underwent SLN biopsy and had their SLNs examined intra-operatively with the OSNA assay, 161 with SLN metastasis and treated with completion axillary lymph node dissection (cALND) were randomly divided into a training (n = 81) and a validation (n = 80) cohort. Non-SLN metastasis of the training cohort was associated with the number of positive SLNs (P = 0.001), CK19 mRNA copy number (P = 0.001), and clinical tumor size (P = 0.055). These parameters were used to construct the intra-operative prediction model of non-SLN metastasis. Its diagnostic accuracy (AUC of ROC curve) was 0.809 and 0.704 for the training and validation cohorts, respectively. The intra-operative prediction model using OSNA may have a diagnostic accuracy of non-SLN metastasis comparable to that of the conventional, post-operative prediction model, indicating that it might help decide the indication for cALND.

Correlation of Methylated Circulating Tumor DNA With Response to Neoadjuvant Chemotherapy in Breast Cancer Patients.

Micro‐Abstract Correlation of methylated circulating tumor DNA (met‐ctDNA) with tumor response to neoadjuvant chemotherapy (NAC) was evaluated in breast cancer patients. In patients with positive met‐ctDNA before NAC, met‐ctDNA significantly correlated with tumor response and was found to be a more sensitive marker than carcinoembryonic antigen and cancer‐associated antigen 15‐3. The possibility has also been suggested that met‐ctDNA might be useful in monitoring the postoperative recurrence. Background: Circulating tumor DNA (ctDNA) is known to harbor tumor‐specific genetic or epigenetic alterations. In the present study, the correlation of ctDNA with tumor response to neoadjuvant chemotherapy (NAC) was evaluated in primary breast cancer patients. Patients and Methods: Plasma samples were obtained from 87 primary breast cancer patients (stage II‐III) before and after NAC, as well as 1 year after surgery. Methylated ctDNA (met‐ctDNA) was determined by one‐step methylation‐specific PCR (OS‐MSP) for the promoter region of RASSF1A. Results: The positivity (23.0%, 20/87) of met‐ctDNA before NAC was significantly (P < .05) higher than that of carcinoembryonic antigen (CEA) (8.6%) and cancer‐associated antigen (CA) 15‐3 (7.4%). In the patients with positive met‐ctDNA before NAC, met‐ctDNA significantly decreased after NAC in those with disease that responded to therapy (P = .006), but not in patients whose disease did not respond to therapy. Met‐ctDNA after NAC was found to be significantly (P = .008) correlated to the extent of residual tumor burden. Of the 7 patients who showed an increase in met‐ctDNA at 1 year after surgery, 3 developed recurrence. Conclusion: Met‐ctDNA is a more sensitive marker than CEA and CA15‐3, and it might be useful in monitoring the clinical tumor response to NAC. In addition, the potential use of met‐ctDNA as a tumor marker for monitoring postoperative recurrence has been suggested.

Prediction of pathological complete response to neoadjuvant chemotherapy by magnetic resonance imaging in breast cancer patients

The purpose of this study was to evaluate whether the baseline breast MRI findings would be useful for the prediction for pathological complete response (pCR) by breast cancer patients to neoadjuvant chemotherapy. Primary breast cancer patients (stage II-III) preoperatively treated with sequential paclitaxel (12 cycles) and fluorouracil, epirubicin, and cyclophosphamide (4 cycles), followed by surgery were retrospectively enrolled, and 229 patients were eligible. Before chemotherapy, breast MRI studies were performed. Breast tumors were dichotomized into round + oval and irregular types based on MRI morphology. The round + oval tumors showed a significantly higher pCR rate than the irregular tumors (42.0% vs 17.3%; P < 0.001). In addition, PAM50 analysis revealed that basal and HER2-enriched tumors were significantly more prevalent among round + oval than irregular type tumors (P = 0.015). Baseline MRI morphology appears to be a significant predictor for pCR. The higher rate of the basal and HER2-enriched tumors among the round + oval tumors may explain their better chemo-sensitivity.