Mototsugu Watanabe

REIC/Dkk-3 overexpression downregulates P-glycoprotein in multidrug-resistant MCF7/ADR cells and induces apoptosis in breast cancer

The overexpression of reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), a tumor suppressor gene, induced apoptosis in human prostatic and testicular cancer cells. The aim of this study is to examine the potential of REIC/Dkk-3 as a therapeutic target against breast cancer. First, the in vitro apoptotic effect of Ad-REIC treatment was investigated in breast cancer cell lines and the adenovirus-mediated overexpression of REIC/Dkk-3 was thus found to lead to apoptotic cell death in a c-Jun-NH2-kinase (JNK) phosphorylaion-dependent manner. Moreover, an in vivo apoptotic effect and MCF/Wt tumor growth inhibition were observed in the mouse model after intratumoral Ad-REIC injection. As multidrug resistance (MDR) is a major problem in the chemotherapy of progressive breast cancer, the in vitro effects of Ad-REIC treatment were investigated in terms of the sensitivity of multidrug-resistant MCF7/ADR cells to doxorubicin and of the P-glycoprotein expression. Ad-REIC treatment in MCF7/ADR cells also downregulated P-glycoprotein expresssion through JNK activation, and sensitized its drug resistance against doxorubicin. Therefore, not only apoptosis induction but also the reversal of anticancer drug resistance was achieved using Ad-REIC. We suggest that REIC/Dkk-3 is a novel target for breast cancer treatment and that Ad-REIC might be an attractive agent against drug-resistant cancer in combination with conventional antineoplastic agents.

Adenovirus-mediated REIC/Dkk-3 gene transfer inhibits tumor growth and metastasis in an orthotopic prostate cancer model.

We had previously reported that REIC/Dkk-3, a member of the Dickkopf (Dkk) gene family, works as a tumor suppressor. In this study, we evaluated the therapeutic effects of an intratumoral injection with adenoviral vector encoding REIC/Dkk-3 gene (Ad-REIC) using an orthotopic mouse prostate cancer model of RM-9 cells. We also investigated the in vivo anti-metastatic effect and in vitro anti-invasion effect of Ad-REIC gene delivery. We demonstrated that the Ad-REIC treatment inhibited prostate cancer growth and lymph node metastasis, and prolonged mice survival in the model. These therapeutic responses were consistent with the intratumoral apoptosis induction and in vitro suppression of cell invasion/migration with reduced matrix metalloprotease-2 activity. We thus concluded that in situ Ad-REIC/Dkk-3 gene transfer may be a promising therapeutic intervention modality for the treatment of prostate cancer.

Direct and distant antitumor effects of a telomerase-selective oncolytic adenoviral agent, OBP-301, in a mouse prostate cancer model

We previously constructed OBP-301 (Telomelysin, a telomerase-specific replication-competent adenovirus with human telomerase reverse transcriptase (hTERT) promoter), which showed a strong anticancer effect by inducing cell lysis of human non-small cell lung cancer and colorectal cancer cells. To investigate the utility of OBP-301 for prostate cancer treatment, we herein evaluate the cell killing and antitumor effects. First, in vitro hTERT-specific adenovirus transduction in human prostate cancer cells (LNCaP, PC3, DU145) was confirmed using OBP-401 (Telomelysin-green fluorescent protein (GFP)). There was no detectable GFP transduction in the human prostate normal cells (PrEC, PrSC). Consistently, the cell-killing effect of OBP-301 was observed only in the cancer cells. Second, using an in vivo subcutaneous LNCaP tumor model in nude mice, we demonstrated that three intratumoral OBP-301 injections (107 PFU per tumor × 3 days) were sufficient to eradicate the detectable LNCaP prostate tumor. We also demonstrated that the ispilateral treatment with OBP-301 significantly suppressed contralateral LNCaP tumor growth in both sides of the tumor model. Histological and immunohistochemical analyses revealed diffuse oncolytic degeneration and adenoviral E1A protein expression in both sides of the tumors. Therefore, in situ OBP-301 administration could be a promising therapeutic strategy against prostate cancer and its metastatic lesions.

Antitumor effect of afatinib, as a human epidermal growth factor receptor 2‐targeted therapy, in lung cancers harboring HER2 oncogene alterations

Human epidermal growth factor receptor 2 (HER2) is a member of the HER family of proteins containing four receptor tyrosine kinases. It plays an important role in the pathogenesis of certain human cancers. In non‐small‐cell lung cancer (NSCLC), HER2 amplification or mutations have been reported. However, little is known about the benefit of HER2‐targeted therapy for NSCLCs harboring HER2 alterations. In this study, we investigated the antitumor effect of afatinib, an irreversible epidermal growth factor receptor (EGFR)–HER2 dual inhibitor, in lung cancers harboring HER2 oncogene alterations, including novel HER2 mutations in the transmembrane domain, which we recently identified. Normal bronchial epithelial cells, BEAS‐2B, ectopically overexpressing wild‐type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, and G660D) showed constitutive autophosphorylation of HER2 and activation of downstream signaling. They were sensitive to afatinib, but insensitive to gefitinib. Furthermore, we examined the antitumor activity of afatinib and gefitinib in several NSCLC cell lines, and investigated the association between their genetic alterations and sensitivity to afatinib treatment. In HER2‐altered NSCLC cells (H2170, Calu‐3, and H1781), afatinib downregulated the phosphorylation of HER2 and EGFR as well as their downstream signaling, and induced an antiproliferative effect through G1 arrest and apoptotic cell death. In contrast, HER2‐ or EGFR‐non‐dependent NSCLC cells were insensitive to afatinib. In addition, these effects were confirmed in vivo by using a xenograft mouse model of HER2‐altered lung cancer cells. Our results suggest that afatinib is a therapeutic option as a HER2‐targeted therapy for NSCLC harboring HER2 amplification or mutations.

Acquisition of cancer stem cell-like properties in non-small cell lung cancer with acquired resistance to afatinib

Afatinib is an irreversible epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor (TKI) that is known to be effective against the EGFR T790M variant, which accounts for half of the mechanisms of acquired resistance to reversible EGFR‐TKIs. However, acquired resistance to afatinib was also observed in clinical use. Thus, elucidating and overcoming the mechanisms of resistance are important issues in the treatment of non‐small cell lung cancer. In this study, we established various afatinib‐resistant cell lines and investigated the resistance mechanisms. EGFR T790M mutations were not detected using direct sequencing in established resistant cells. Several afatinib‐resistant cell lines displayed MET amplification, and these cells were sensitive to the combination of afatinib plus crizotinib. As a further investigation, a cell line that acquired resistance to afatinib plus crizotinib, HCC827‐ACR, was established from one of the MET amplified‐cell lines. Several afatinib‐resistant cell lines including HCC827‐ACR displayed epithelial‐to‐mesenchymal transition (EMT) features and epigenetic silencing of miR‐200c, which is a suppresser of EMT. In addition, these cell lines also exhibited overexpression of ALDH1A1 and ABCB1, which are putative stem cell markers, and resistance to docetaxel. In conclusion, we established afatinib‐resistant cells and found that MET amplification, EMT, and stem cell‐like features are observed in cells with acquired resistance to EGFR‐TKIs. This finding may provide clues to overcoming resistance to EGFR‐TKIs.

Targeting the miR-200c/LIN28B axis in acquired EGFR-TKI resistance non-small cell lung cancer cells harboring EMT features.

MicroRNA (miR)-200 family members (miR-200s) are frequently silenced in advanced cancer and have been implicated in the process of epithelial-to-mesenchymal transition (EMT). We previously reported that miR-200s were silenced through promoter methylation in acquired EGFR-tyrosine kinase inhibitor (TKI) resistant non-small cell lung cancer (NSCLC) cells harboring EMT features. In this study, we examined the functional role of miR-200s in NSCLC cells and investigated a novel approach to overcoming acquired EGFR-TKI resistance. In the analysis of NSCLC cell lines, each of the miR-200s expression-silenced cell lines showed promoter methylation. Significant correlations between miR-200c silencing and several oncogenic pathway alterations, including EMT-changes and LIN28B overexpression, were observed in the database analysis. In addition, EGFR-wild type cell lines had lower miR-200s expression levels than EGFR-mutant cell lines. The introduction of miR-200c using pre-miR-200c caused LIN28B suppression in cells with acquired EGFR-TKI resistance that harbored EMT features. Interestingly, both the introduction of miR-200c and the knockdown of LIN28B produced an antitumor effect in acquired EGFR-TKI resistance cells, whereas these manipulations were not effective in parental cells. The miR-200c/LIN28B axis plays an important role in cells with acquired resistance to EGFR-TKI that harbor EMT features and might be a useful therapeutic target for overcoming resistance.

Interaction of cytokeratin 19 head domain and HER2 in the cytoplasm leads to activation of HER2-Erk pathway

HER2 is a receptor tyrosine kinase and its upregulation via activating mutations or amplification has been identified in some malignant tumors, including lung cancers. Because HER2 can be a therapeutic target in HER2-driven malignancies, it is important to understand the molecular mechanisms of HER2 activation. In the current study, we identified that cytokeratin 19 (KRT19) binds to HER2 at the inside face of plasma membrane. HER2 and KRT19, which were concurrently introduced to a human embryonic kidney 293 T cells, revealed an association with each other and resulted in phosphorylation of HER2 with the subsequent activation of a downstream Erk-associated pathway. A binding assay revealed that both the NH2-terminal head domain of KRT19 and the COOH-terminal domain of HER2 were essential for their binding. To investigate the impact of the interaction between HER2 and KRT19 in lung cancer, we examined their expressions and localizations in lung cancers. We found that KRT19 was highly expressed in HER2-positive lung cancer cells, and KRT19 and HER2 were co-localized at the cell membrane. In conclusion, we found that KRT19 intracellularly binds to HER2, playing a critical role in HER2 activation.

Yes1 signaling mediates the resistance to Trastuzumab/Lap atinib in breast cancer.

Background Overexpression of human epidermal growth factor receptor 2 (HER2) is observed in approximately 15–23% of breast cancers and these cancers are classified as HER2-positive breast cancer. Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer and has improved patient overall survival. However, acquired resistance to trastuzumab is still a critical issue in breast cancer treatment. We previously established a trastuzumab-resistant breast cancer cell line (named as BT-474-R) from a trastuzumab-sensitive HER2-amplified cell line BT-474. Lapatinib is also a molecular-targeted drug for HER2-positive breast cancer, which acquired the resistance to trastuzumab. Acquired resistance to lapatinib is also an issue to be conquered. Methods We established trastuzumab/lapatinib-dual resistant cell line (named as BT-474-RL2) by additionally treating BT-474-R with lapatinib. We analyzed the mechanisms of resistance to trastuzumab and lapatinib. Besides, we analyzed the effect of the detected resistance mechanism in HER2-positive breast cancer patients. Results Proto-oncogene tyrosine-protein kinase Yes1, which is one of the Src family members, was amplified, overexpressed and activated in BT-474-R and BT-474-RL2. Silencing of Yes1 by siRNA induced both BT-474-R and BT-474-RL2 to restore the sensitivity to trastuzumab and lapatinib. Pharmaceutical inhibition of Yes1 by the Src inhibitor dasatinib was also effective to restore the sensitivity to trastuzumab and lapatinib in the two resistant cell lines. Combination treatment with dasatinib and trastuzumab induced down-regulation of signaling molecules such as HER2 and Akt. Moreover, the combination treatments induced G1-phase cell-cycle arrest and apoptosis. Consistent with cell line data, high expression of Yes1 mRNA was correlated with worse prognosis in patients with HER2-positive breast cancer. Conclusion Yes1 plays an important role in acquired resistance to trastuzumab and lapatinib in HER2-positive breast cancer. Our data suggest that pharmacological inhibition of Yes1 may be an effective strategy to overcome resistance to trastuzumab and lapatinib.

Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods

Techniques for the extraction and use of nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissues, preserved over long time periods in libraries, have been developed. However, DNA extracted from FFPE tissues is generally damaged, and long-term storage may affect DNA quality. Therefore, it is important to elucidate the effect of long-term storage on FFPE tissues and evaluate the techniques used to extract DNA from them. In the present study, the yield, purity, and integrity of DNA in FFPE tissue samples was evaluated. Two DNA extraction techniques were used: A silica-binding DNA collection method using QIAamp DNA FFPE Tissue kit (QIA) and a total tissue DNA collection method using a WaxFree DNA extraction kit (WAX). A total of 25 FFPE tissues from lung adenocarcinomas were studied, which had been surgically resected and fixed at Okayama University Hospital prior to examination and subsequent storage at room temperature for 0.5, 3, 6, 9 and 12 years. Extracted DNA was quantified using ultraviolet absorbance, fluorescent dye, and quantitative polymerase chain reaction (qPCR). The quality of the DNA was defined by the absorbance ratio of 260 to 280 nm (A260/280) and Q-score, which is the quantitative value of qPCR product size ratio. The results demonstrated that the yield of total DNA extracted using WAX was significantly greater than when QIA was used (P<0.01); however, DNA extracted using WAX included more contaminants and was significantly more fragmented compared with DNA extracted using QIA (P<0.01). Aging had no significant effect on absolute DNA yield or DNA purity, although it did significantly contribute to increased DNA degradation for both QIA and WAX extraction (QIA P=0.02, WAX P=0.03; 0.5 years vs. 3 years, QIA P<0.01, WAX P=0.03; 9 years vs. 12 years). Both extraction methods are viable depending on whether high yield or high quality of extracted DNA is required. However, due to the increased degradation with age, storage time limits the available DNA in FFPE tissues regardless of the extraction method.

Elacridar, a third-generation ABCB1 inhibitor, overcomes resistance to docetaxel in non‑small cell lung cancer

Docetaxel is a third-generation chemotherapeutic drug that is widely used in the treatment of patients with non-small cell lung cancer (NSCLC). However, the majority of patients with NSCLC eventually acquire resistance to the treatment. In the present study, the mechanism of acquired resistance to docetaxel treatment in lung cancer cells was investigated. The three NSCLC cell lines, H1299 with wild-type epidermal growth factor receptor (EGFR), EGFR-mutant HCC4006 and HCC827, and experimentally established docetaxel-resistant (DR) cells, H1299-DR, HCC827-DR, and HCC4006-DR were used with stepwise increases in concentrations of docetaxel. It was demonstrated that the established cell lines showed resistance to docetaxel and EGFR-tyrosine kinase inhibitors (TKIs). Molecular analysis revealed that all of the resistant cell lines highly expressed ATP binding cassette subfamily B member 1 (ABCB1), which is also known as P-glycoprotein or MDR1. Furthermore, HCC827-DR and HCC4006-DR cells exhibited a cancer stem cell-like marker and epithelial-to-mesenchymal transition features, respectively. Elacridar (GF120918), a third-generation inhibitor of ABCB1, was able to overcome resistance to docetaxel. Additionally, knockdown of ABCB1 using small interfering RNA (si)-ABCB1 recovered sensitivity to docetaxel. However, elacridar and si-ABCB1 could not recover sensitivity to EGFR-TKIs in established resistant cells. The results of the present study revealed that docetaxel-resistant NSCLC cells also acquired cross-resistance to EGFR-TKI therapy through mechanisms other than ABCB1, that ABCB1 serves an important role in acquired resistance to docetaxel in lung cancer, and that combination therapy with elacridar can overcome ABCB1-mediated docetaxel resistance.