Masaya Kai

Hypoxia activates the hedgehog signaling pathway in a ligand-independent manner by upregulation of Smo transcription in pancreatic cancer

The hedgehog (Hh) signaling pathway is activated in various types of cancer including pancreatic ductal adenocarcinoma. It has been shown that extremely low oxygen tension (below 1% O2) is found in tumor tissue including pancreatic ductal adenocarcinoma cells (PDAC) and increases the invasiveness of PDAC. To investigate the contribution of the Hh pathway to hypoxia‐induced invasiveness, we examined how hypoxia affects Hh pathway activation and the invasiveness of PDAC. In the present study, three human PDAC lines were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions. Hypoxia upregulated the transcription of Sonic hedgehog (Shh), Smoothened (Smo), Gli1 and matrix metalloproteinase9 (MMP9) and increased the invasiveness of PDAC. Significantly, neither the addition of recombinant Shh (rhShh) nor the silencing of Shh affected the transcription of these genes and the invasiveness of PDAC. On the other hand, silencing of Smo decreased the transcription of Gli1 and MMP9 and PDAC invasiveness. Silencing of Gli1 or MMP9 decreased PDAC invasiveness. These results suggest that hypoxia activates the Hh pathway of PDAC by increasing the transcription of Smo in a ligand‐independent manner and increases PDAC invasiveness. (Cancer Sci 2011; 102: 1144–1150)

Hedgehog signaling pathway mediates the progression of non‐invasive breast cancer to invasive breast cancer

The purpose of this study is to clarify the contribution of the Hedgehog signaling pathway (Hh pathway) to the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). A total of 149 surgically resected mammary disease specimens and 12 sentinel lymph nodes with micro‐metastasis (Ly‐met) were studied. The degree of Hh pathway activation was estimated from the Gli1 nuclear staining ratio (%Gli1 nuclear translocation) in cancer cells. The invasiveness of breast cancer cells was determined using Matrigel assays. A serial increase of %Gli1 nuclear translocation to IDC from non‐neoplastic diseases was confirmed. In tumor specimens, %Gli1 nuclear translocation correlated with the invasiveness of each type of mammary disease and also correlated with invasion‐related histopathological parameters. The %Gli1 nuclear translocation in lymph nodes with micro‐metastasis was similar to that in primary sites and higher than that in DCIS with microinvasion and DCIS. Blockade of the Hh pathway decreased the invasiveness of breast cancer cells. In IDC, %Gli1 nuclear translocation correlated with the expression of estrogen receptor‐α. Estrogen increased %Gli1 nuclear translocation and the invasiveness of estrogen receptor‐α‐positive cells. The Hh pathway mediates progression from a non‐invasive phenotype to an invasive phenotype and %Gli1 nuclear translocation may be useful as a predictive marker for evaluating the ability of invasiveness. (Cancer Sci 2011; 102: 373–381)

Hedgehog inhibitor decreases chemosensitivity to 5‐fluorouracil and gemcitabine under hypoxic conditions in pancreatic cancer

Pancreatic cancer is one of the deadliest types of cancer. Previously, we showed that hypoxia increases invasiveness through upregulation of Smoothened (Smo) transcription in pancreatic ductal adenocarcinoma (PDAC) cells. Here, we first evaluated whether hypoxia‐induced increase in Smo contributes to the proliferation of PDAC cells. We showed that Smo, but not Gli1, inhibition decreases proliferation significantly under hypoxic conditions. To further investigate the effects of Smo on PDAC growth, cell cycle analysis was carried out. Inhibition of Smo under hypoxia led to G0/G1 arrest and decreased S phase. As 5‐fluorouracil (5‐FU) and gemcitabine, which are first‐line drugs for pancreatic cancer, are sensitive to S phase, we then evaluated whether cyclopamine‐induced decreased S phase under hypoxia affected the chemosensitivity of 5‐FU and gemcitabine in PDAC cells. Cyclopamine treatment under hypoxia significantly decreased chemosensitivity to 5‐FU and gemcitabine under hypoxia in both in vitro and in vivo models. In contrast, cis‐diamminedichloroplatinum, which is cell cycle‐independent, showed significant synergistic effects. These results suggest that hypoxia‐induced increase of Smo directly contributes to the proliferation of PDAC cells through a hedgehog/Gli1‐independent pathway, and that decreased S phase due to the use of Smo inhibitor under hypoxia leads to chemoresistance in S phase‐sensitive anticancer drugs. Our results could be very important clinically because a clinical trial using Smo inhibitors and chemotherapy drugs will begin in the near future. (Cancer Sci 2012; 103: 1272–1279)

The combination of PD-L1 expression and decreased tumor-infiltrating lymphocytes is associated with a poor prognosis in triple-negative breast cancer

This study included patients with primary triple-negative breast cancer (TNBC) who underwent resection without neoadjuvant chemotherapy between January 2004 and December 2014. Among the 248 TNBCs studied, programmed cell death ligand-1 (PD-L1) expression was detected in 103 (41.5%) tumors, and high levels of tumor-infiltrating lymphocytes (TILs) were present in 118 (47.6%) tumors. PD-L1 expression correlated with high levels of TILs, but was not a prognostic factor. Patients with TILs-high tumors had better overall survival than those with TILs-low tumors (P = 0.016). There was a strong interaction between PD-L1 expression and TILs that was associated with both recurrence-free survival (P = 0.0018) and overall survival (P = 0.015). Multivariate Cox proportional hazards model analysis showed that PD-L1-positive/TILs-low was an independent negative prognostic factor for both recurrence-free survival and overall survival. Our findings suggest that PD-L1-positive/TILs-low tumors are associated with a poor prognosis in patients with TNBC, and that it is important to focus on the combination of PD-L1 expression on tumor cells and TILs present in the tumor microenvironment. These biomarkers may be useful for stratification of TNBCs and for predicting prognosis and developing novel cancer immunotherapies.

Semi-quantitative evaluation of CD44+/CD24− tumor cell distribution in breast cancer tissue using a newly developed fluorescence immunohistochemical staining method

CD44+/CD24− tumor cells are reported to contain cancer stem cells in breast cancer. The main purpose of the present study is to develop an immunohistofluorescence method that can quantitatively analyze CD44+/CD24− tumor cell distribution in breast cancer tissue and help better define the role of CD44+/CD24− tumor cells in breast cancer. The samples used were from 21 primary breast cancer patients who underwent neoadjuvant chemotherapy and 17 cases with sentinel lymph nodes that had lymph node micrometastasis. CD44+/CD24− tumor cells were distinguished at a single cell level using improved triple‐staining immunohistofluorescence and a simulated laser capture microdissection method. The percentage of CD44+/CD24− cells significantly increased following neoadjuvant chemotherapy treatment (0.93% and 2.78%, before and after, respectively, P = 0.0043). The percentage of CD44+/CD24− cells was also significantly high in micrometastatic sentinel lymph nodes (0.49% and 1.91%, primary tumors and lymph nodes, respectively, P = 0.0246). The CD44+/CD24− tumor cell distribution was heterogeneous in both breast cancer tissue and lymph node metastasis. In a xenograft model using immunodeficient mice, the hedgehog signaling inhibitor cyclopamine repressed the tumorigenicity of CD44+/CD24− cells. Our results suggest that this semi‐quantitative immunohistochemical analysis is valuable for detecting a small population of cells in cancer tissues and that the hedgehog signaling pathway inhibitor cyclopamine is useful for regulating the CD44+/CD24− tumor cells in breast cancer. (Cancer Sci 2011; 102: 2132–2138)

BRCAness as a Biomarker for Predicting Prognosis and Response to Anthracycline-Based Adjuvant Chemotherapy for Patients with Triple-Negative Breast Cancer.

Background Triple-negative breast cancer (TNBC) is a heterogeneous tumor that encompasses many different subclasses of the disease. In this study, we assessed BRCAness, defined as the shared characteristics between sporadic and BRCA1-mutated tumors, in a large cohort of TNBC cases. Methods The BRCAness of 262 patients with primary TNBCs resected between January 2004 and December 2014 was determined through the isolation of DNA from tumor tissue. Classification of BRCAness was performed using multiple ligation-dependent probe amplification (MLPA). The tumor subtypes were determined immunohistochemically using resected specimens. Results Of the 262 TNBCs, the results of the MLPA assays showed that 174 (66.4%) tumors had BRCAness. Patients with BRCAness tumors were younger than patients with non-BRCAness tumors (P = 0.003). There was no significant difference between the two groups regarding their pathological stages. The BRCAness group had a significantly shorter recurrence-free survival (RFS) compared with the non-BRCAness group (P = 0.04) and had a shorter overall survival (OS) although this did not reach statistical significance. Adjuvant treatments with anthracycline-based regimens provided significantly greater benefits to the BRCAness group (P = 0.003 for RFS, and P = 0.03 for OS). Multivariate Cox proportional hazard model analysis showed that BRCAness was an independent negative prognostic factor, and the anthracycline-based adjuvant chemotherapy was an independent positive prognostic factor for both RFS and OS in TNBC. Conclusions The 66.4% patients of TNBCs showed BRCAness. BRCAness is essential as a biomarker in the subclassification of TNBCs and might be of use for predicting their prognosis. Furthermore, this biomarker might be a predictive factor for the effectiveness of anthracycline-based adjuvant chemotherapy for patients with TNBCs.

BRCAness Combined With a Family History of Cancer Is Associated With a Poor Prognosis for Breast Cancer Patients With a High Risk of BRCA Mutations

Background The inexpensive prediction of the characteristics of BRCA‐mutated breast cancer as “BRCAness” using the somatic cells of patients with breast cancer could be useful for developing a therapeutic strategy. Our objective was to correlate BRCAness with the clinicopathologic features, including a family history (FH) of cancer, in breast cancer patients with a high risk of BRCA mutations. Patients and Methods The present study included 124 patients, including 55 with early‐onset and 77 with triple‐negative breast cancer, who had undergone resection at Kyushu University Hospital from 2005 to 2014. Early‐onset breast cancer is defined as an onset in patients aged ≤ 40 years. BRCAness was performed using multiple ligation‐dependent probe amplification. The patients’ FH of cancer was surveyed from first‐ to third‐degree relatives. Results Of the 124 patients, the multiple ligation‐dependent probe amplification assay results indicated that 59 tumors (47.6%) had BRCAness and 27 patients (21.8%) had a positive FH for cancer. The patients with BRCAness experienced significantly shorter recurrence‐free survival (RFS) and overall survival (OS) compared with those without. Patients with FH had shorter RFS and OS compared to those without BRCAness. The patients were divided into those with and without BRCAness and those with and without a positive FH. The BRCAness with FH subgroup experienced significantly shorter RFS and OS. Multivariate analysis revealed that BRCAness and a positive FH were independent negative prognostic factors. Conclusion Our findings suggest that BRCAness tumors with a positive FH of cancer were associated with a poor prognosis in the BRCA‐mutation high‐risk group. We propose that BRCAness and a positive FH will serve to predict patients’ prognosis. Micro‐Abstract We assessed BRCAness as a characteristic of BRCA‐mutated breast cancer. We investigated 124 breast cancer patients with a high risk of BRCA mutations, who had early‐onset or triple‐negative breast cancer. No correlation was found between BRCAness and family history. However, those with a BRCAness tumor and a positive family history had a significantly worse prognosis, which could be of use in predicting the prognosis.

QOL-enhancing surgery for patients with HER2-positive metastatic breast cancer

The role of the resection of primary tumour in stage IV breast cancer is unclear. Systemic therapy is recommended to prolong the survival and improve the quality of life (QOL). However, even if the systemic therapy is effective to control distant metastasis, sometimes the local lesion worsens, especially in the aggressive subtypes such as HER2-positive breast cancer. In uncontrollable tumours, the wound bed can bleed, weep and get infected, leading to dismal QOL. Our study describes two cases of patients with HER2-positive stage IV breast cancer who underwent palliative mastectomy which resulted in improvement of QOL. Local tumour control through palliative mastectomy can be beneficial for symptomatic aggressive patients with HER2-positive breast cancer to improve their QOL.

Efficacy and safety of bi-weekly pegfilgrastim for dose-dense chemotherapy-induced neutropenia in breast cancer patients

Background/Aim: The dose-dense doxorubicin and cyclophosphamide (ddAC) for patients with HER-2-negative breast cancer is recommended by the National Comprehensive Cancer Network guideline in US. However, there are little data on serum G-CSF concentrations in patients undergoing bi-weekly dose-dense therapy with pegfilgrastim. The objective of this study was to compare the serum G-CSF concentrations in patients receiving pegfilgrastim in bi- or tri-weekly regimens. Patients and Methods: This study included 26 patients who received ddAC or docetaxel and cyclophosphamide (TC) for primary breast cancer. Serum G-CSF concentrations were measured by ELISA. Results: Serum G-CSF concentrations peaked in the second week of ddAC cases and in the ninth week of TC cases. Neutrophils gradually increased until the sixth week in ddAC cases, while they were slightly decreased during the first three weeks in TC cases. Treatments were completed without febrile neutropenia or treatment delays. Conclusion: Primary prophylactic pegfilgrastim administrations increased serum G-CSF concentrations, helping to maintain the absolute neutrophil counts that are required to undergo chemotherapy. The treatment of ddAC with 3.6 mg pegfilgrastim is completely safe for female Japanese patients.

Visualizing energy charge in breast carcinoma tissues by MALDI mass-spectrometry imaging profiles of low-molecular-weight metabolites

Background/Aim: Metabolomics is widely used for biomarker discovery, but conventional mass-spectrometry extraction procedures lose the spatial localization of metabolites. In this study, we directly analyzed breast carcinoma tissues embedded in frozen tissue microarrays (fTMAs) using MALDI mass-spectrometry imaging (MALDI-MSI). Materials and Methods: A total of 119 breast tissues (84 carcinoma and 35 normal) were used. MSI data were extracted from each tissue. Results: Overall, 185 of 1,915 peaks which were commonly detected in 60% of target areas were subjected to further analysis. One hundred and fifty-two peaks of carcinoma showed significantly higher intensity than normal. Comparing metabolite profiles from carcinoma and normal tissues, energy charge (EC) and the sum of adenosine phosphate compound (AXP) indicated significantly higher intensities in cancerous tissues than normal. But comparisons of EC and AXP among lymph node metastasis, tumor size and tumor subtypes indicated no significant differences. Conclusion: Breast carcinoma tissues had higher EC and AXP values than normal. MALDI-MSI could be a tool for characterizing breast carcinoma.