Hiroyoshi Doihara

Role of epidermal growth factor receptor in breast cancer

Decades of research in molecular oncology have brought about promising new therapies which are designed to target specific molecules which promote tumor growth and survival. The epidermal growth factor receptor (EGFR) is one of the first identified important targets of these novel antitumor agents. Approximately half of cases of triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC) overexpress EGFR. Thus, EGFR inhibitors for treatment of breast cancer have been evaluated in several studies. However, results so far have been disappointing. One of the reasons for these unexpected results is the lack of biomarkers for predicting which patients are most likely to respond to EGFR inhibitors. Recent studies have shown that EGFR and its downstream pathway regulate epithelial-mesenchymal transition, migration, and tumor invasion and that high EGFR expression is an independent predictor of poor prognosis in IBC. Further, recent studies have shown that targeting EGFR enhances the chemosensitivity of TNBC cells by rewiring apoptotic signaling networks in TNBC. These studies indicate that EGFR-targeted therapy might have a promising role in TNBC and IBC. Further studies of the role of EGFR in TNBC and IBC are needed to better understand the best way to use EGFR-targeted therapy—e.g., as a chemosensitizer or to prevent metastases—to treat these aggressive diseases.

PTEN activity could be a predictive marker of trastuzumab efficacy in the treatment of ErbB2-overexpressing breast cancer

Trastuzumab is the only HER2/neu-directed therapy to have received Food and Drug Administration approval for the treatment of patients with metastatic breast cancer. The efficacy of trastuzumab depends on the HER2/neu status of the tumour and the patients prior treatment, but even when patients are selected on the basis of HER2/neu gene amplification, the single-agent response rate ranges from 12 to 30% and few patients respond to trastuzumab monotherapy. Here, we propose PTEN as a predictive biomarker for trastuzumab efficacy. Human breast cancer SKBR3 and drug-resistant SKBR3/R cells were investigated. We also examined clinical samples from patients who had been treated with trastuzumab and analysed the relationship between trastuzumab efficacy and PTEN level. The PI3K/Akt signalling pathway was observed to be highly active in the drug-resistant cells, and their level of PTEN was low. Delivery of antisense PTEN duplex siRNA significantly decreased the trastuzumab chemosensitivity of parental SKBR3 cells, and marked activation of Akt signalling pathway was also recognised. Moreover, immunohistochemical investigation revealed that trastuzumab treatment was remarkably successful in cells with elevated PTEN expression. Along with the immune-system-associated cytotoxic mechanism, several mechanisms have been proposed for the effect of trastuzumab. PTEN activity might play an important and major role in its HER2/PI3K/Akt-mediated antitumour effect, and could be a useful biomarker for predicting the efficacy of trastuzumab in the treatment of breast cancer.

Aberrant Promoter Methylation in Human DAB2 Interactive Protein (hDAB2IP) Gene in Breast Cancer

Purpose: Human DOC-2/DAB2 interactive protein (hDAB2IP) gene is a novel member of the Ras GTPase-activating family and has been demonstrated to be a tumor suppressor gene inactivated by methylation in prostate cancer. We analyzed methylation and expression status of hDAB2IP in breast cancer. Experimental Design: The promoter region of hDAB2IP was divided into two regions (m2a and m2b) following our previous report on prostate cancer, and methylation status was determined in breast cancer cell lines with bisulfited DNA sequencing. Expression was semiquantified with real-time reverse transcription-PCR to find that aberrant methylation showed the inverse relationship with expression. On the basis of sequence data, we developed methylation-specific PCR for m2a and m2b regions and applied to samples. Results: Aberrant methylation was detected in 11 of 25 breast cancer cell lines (44%) and 15 of 39 primary tumors (38%) at the m2a region and in 12 of 25 cell lines (48%) and 13 of 39 tumors (33%) at the m2b region. In addition, gene expression was restored in methylated cell lines with 5-aza-2′-deoxycytidine, confirming that methylation caused gene down-regulation. We also examined the relationship between hDAB2IP methylation and clinicopathologic features in primary tumors and found that methylation in the m2b region was associated with progressive nodal status of tumors. Conclusions: We developed methylation-specific PCR for hDAB2IP and examined its methylation status in breast cancer. Our results demonstrate that hDAB2IP methylation frequently is present in breast cancer and plays a key role in hDAB2IP inactivation, suggesting the relationship between hDAB2IP methylation and lymph node metastasis of breast cancer.

nm23-H1 reduces in vitro cell migration and the liver metastatic potential of colon cancer cells by regulating myosin light chain phosphorylation.

The nm23‐H1 gene is known as a potential metastasis suppressor gene in various types of carcinomas. However, the role of nm23‐H1 in colorectal carcinoma still remains controversial and the cellular mechanisms by which its protein may modulate the metastatic phenotype are not yet known. We transfected nm23‐H1 cDNA into the human colon cancer cell line, HT‐29, to test the effects and cellular biological mechanism of nm23 protein in colon cancer. We found that nm23‐H1 strongly inhibited the liver metastasis of HT‐29 cells in nude mice and inhibited the epidermal growth factor (EGF)‐induced cell migration in vitro. Furthermore, we clarified the regulation of the myosin light chain (MLC) phosphorylation by nm23‐H1, which has been demonstrated as having potential role in cell migration.

Aberrant promoter methylation of insulin‐like growth factor binding protein‐3 gene in human cancers

Insulin‐like growth factor binding protein‐3 (IGFBP‐3) is postulated to be a mediator of growth suppression signals. Here, we examined the methylation status of IGFBP‐3 to correlate to clinicopathological factors in human cancers. The methylation status of IGFBP‐3 was determined by bisulfite DNA sequencing and was correlated with expression semi‐quantified by real‐time RT‐PCR to develop a methylation‐specific PCR (MSP) assay for IGFBP‐3. Using the MSP assay, we examined the methylation status of IGFBP‐3 in gastric cancer (GC), colorectal cancer (CRC), breast cancer (BC) and malignant mesothelioma (MM). IGFBP‐3 methylation was detected in 6 of 13 (46%) and 16 of 24 (67%) GC cell lines and tumors, respectively; 4 of 8 (50%) and 15 of 26 (58%) CRC cell lines and tumors, respectively; 3 of 11 (27%) and 7 of 39 (18%) BC cell lines and tumors, respectively and 1 of 5 (20%) and 18 of 56 (32%) MM cell lines and tumors, respectively. Interestingly, the methylation status of MM specimens from Japanese patients (75%, 12 out of 16 patients) was significantly higher than those from the USA (15%, 6 out of 40 patients) (p < 0.0001), suggesting the presence of ethnic differences in the IGFBP‐3 methylation status. We also found that IGFBP‐3 methylation was preferentially present in GCs arising in the lower‐third of the stomach (p = 0.079). In summary, our results showed that IGFBP‐3 methylation played an important role in the silencing of its expression, suggesting that IGFBP‐3 may act as a tumor suppressor gene in several human cancers examined.

Proteasome inhibitors can alter the signaling pathways and attenuate the P‐glycoprotein‐mediated multidrug resistance

Numerous signaling pathways were reported to be involved in the resistance for conventional cytotoxic drugs, although one of the main reasons is the overexpression of P‐glycoprotein (P‐gp) in multidrug resistant cancer cells. The overexpression of P‐gp has been associated with the resistance to a wide range of anticancer drugs. Doxorubicin and paclitaxel are substrates of this transporter system and have an important role for the various human malignancies. In the present study, drug‐sensitive MCF7 and multidrug resistant MCF7/ADR (characterized by overexpression of P‐gp) human breast cancer cell lines were used as an experimental model. We have found that PS341 and MG132, proteasome inhibitors, reduced the degree of the multidrug resistance (MDR) in MCF7/ADR cells. This phenomenon was accompanied by a decrease in the IC50 value of doxorubicin and paclitaxel from 55.9 ± 3.46 to 0.60 ± 0.08 μM, and from 17.61 ± 1.77 to 0.59 ± 0.12 μM, respectively. The IC50 values of sensitive cells for doxorubicin and paclitaxel were about 0.42 and 0.83 μM, respectively. The effect of PS341 and MG132 on MCF7/ADR cells was associated with a significant decrease in both protein and gene levels of P‐gp expression. Moreover, with regard to the expression of possible signal transduction pathways of mitogen‐activated protein kinase (MAPK) related to the activation of mdr1, proteasome inhibitors did significantly influence the activation of these proteins. Western blot analysis revealed that 24 hr exposure of multidrug resistant MCF7/ADR cells with proteasome inhibitors did change the levels of DNA binding activity of nuclear factor‐kappaB (NF‐kappaB), pERK1/2, c‐Jun, and p‐c‐Jun. In conclusion, we could remark that proteasome inhibitors (especially PS341) attenuate the resistance of MCF7/ADR cells for P‐gp substrate drugs of doxorubicin and paclitaxel. Several proteins are supposed to be associated with the resensitization of the cells to conventional cytotoxic drugs, although decreased activity of P‐gp is at least involved in the proteasome inhibitor‐related resensitization. And influence with MAPK pathways, which have been reported to be associated with the regulation of P‐gp, might be contributed to the resensitization brought by proteasome inhibitors.

Bone morphogenetic protein 6 (BMP6) and BMP7 inhibit estrogen-induced proliferation of breast cancer cells by suppressing p38 mitogen-activated protein kinase activation

Estrogen is involved in the development and progression of breast cancer. Here, we investigated the effects of bone morphogenetic proteins (BMPs) on breast cancer cell proliferation caused by estrogen using human breast cancer MCF-7 cells. MCF-7 cells express estrogen receptors (ESR1 and ESR2), BMP receptors, and SMAD signaling molecules. Estradiol and membrane-impermeable estradiol stimulated MCF-7 cell proliferation. Estradiol also reduced mRNA levels of ESR1, aromatase, and steroid sulfatase. Treatment with BMPs and activin had no effects on MCF-7 cell proliferation. However, BMP2, BMP4, BMP6, BMP7, and activin suppressed estradiol-induced cell mitosis, with the effects of BMP6, BMP7, and activin being more prominent than those of BMP2 and BMP4. Activin decreased ESR1 mRNA expression, while BMP6 and BMP7 impaired steroid sulfatase expression in MCF-7 cells. Interestingly, SMAD1,5,8 activation elicited by BMP6 and BMP7, but not by BMP2 and BMP4, was preserved even under the exposure of a high concentration of estradiol. The difference of BMP responsiveness was likely due to the differential modulation of BMP receptor expression induced by estradiol. In this regard, estradiol decreased the expression levels of BMPR1A, BMPR1B, ACVR2A, and ACVR2B but did not affect ACVR1 and BMPRII, leading to the sustained effects of BMP6 and BMP7 in estrogen-treated MCF-7 cells. Estradiol rapidly activated MAPK phosphorylation including extracellular signal-regulated kinase 1/2, p38, and stress-activated protein kinase/c-Jun NH2-terminal kinase pathways and BMP6, BMP7, and activin preferentially inhibited estradiol-induced p38 phosphorylation. SB203580, a selective p38 MAPK inhibitor effectively suppressed estradiol-induced cell mitosis, suggesting that p38 MAPK plays a key role in estrogen-sensitive breast cancer cell proliferation. Thus, a novel interrelationship between estrogen and the breast cancer BMP system was uncovered, in which inhibitory effects of BMP6 and BMP7 on p38 signaling and steroid sulfatase expression were functionally involved in the suppression of estrogen-induced mitosis of breast cancer cells.

Effects of toremifene (TOR) and tamoxifen (TAM) on serum lipids in postmenopausal patients with breast cancer.

This study clarified the difference in the effects on serum lipids between toremifene (TOR) and tamoxifen (TAM). To remove influencing factors, we investigated adjuvant therapy for hormone receptor-positive patients with breast cancer without lymph node metastasis. The subjects were 65 patients who were enrolled in a multicenter randomized comparative study between April 1997 and March 2001. As adjuvant therapy, 20 mg of TAM or 40 mg of TOR was administered for 1 year. The levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein A-1 (Apo A-1), apolipoprotein A(Apo B), and lipoprotein a (Lp(a)) were measured prior to administration and 3, 6, and 12 months after the start of administration. TC, LDL-C, Lp(a) and Apo B significantly decreased from the third month of administration compared with values before the start of administration in both the TOR and TAM groups. HDL-C significantly increased from the third month only in the TOR group. TG significantly increased in the TAM group but significantly decreased in the TOR group in the 12th month of administration. When these two groups were compared, HDL-C was significantly higher ( p < 0.01) and TG was significantly lower ( p < 0.01) in the TOR group in the 12th month. Improvement of abnormal values of TG, HDL-C and LDL-C was better in the TOR group than in the TAM group after administration for 12 months. The effect on lipid metabolism showed different profiles between the two selective estrogen receptor modulators (SERMs), and TOR gave better results than TAM.

Dissection of the APCCdh1-Skp2 Cascade in Breast Cancer

Purpose: Skp2 is a subunit of the SCF ubiquitin protein ligase, which plays a vital role in the control of tumorigenesis via its regulation of G1-S transition. Deregulation of Skp2 in various types of cancers correlates with aggressive clinical behavior and poor prognosis. Recent studies suggest that cell cycle–dependent fluctuation of Skp2 is governed by APCCdh1, another important E3 ligase, thereby preventing premature entry into S phase. To assess the potential role of APCCdh1 in tumorigenesis through proteolysis of Skp2, we have dissected the APCCdh1-Skp2 cascade. Experimental Design: We manipulated the APCCdh1-Skp2 cascade and examined its cellular behavior using both breast cancer and normal breast epithelial cells. Furthermore, applying immunohistochemistry, we analyzed the clinicopathologic significance of these molecules in patients with breast cancer. Results: Analysis of tissue arrays indicated that the percentage of samples positive for Cdh1 in breast cancer was significantly lower compared with normal breast tissues (P = 0.004). Conversely, the percentage of samples scored as positive for Skp2 in cancer was significantly higher than in normal tissues (P < 0.001). Moreover, prognostic studies revealed that relatively high levels of Cdh1 are associated with survivability in patients with breast cancer. In addition, depletion of Cdh1 by small interfering RNA in normal breast cells resulted in increased cellular proliferation, whereas knockdown of Skp2 significantly suppressed growth in breast cancer cells. Conclusions: This study shows a correlation between Skp2 and APCCdh1 in breast cancer. Thus, Cdh1 may act as an important component in tumor suppression and could be considered as a novel biomarker in breast cancer.

Regulation of Skp2-p27 Axis by the Cdh1/Anaphase-Promoting Complex Pathway in Colorectal Tumorigenesis

Abrogated entry into S phase is a common hallmark of cancer cells. Skp2, a subunit of ubiquitin ligase, is critical for regulating the G(1)/S transition. Uncontrolled Skp2 activity is detected frequently in human tumors, often correlated with poor prognosis. Current studies have suggested that the regulation of Skp2 turnover is mediated by another critical ubiquitin ligase, the anaphase-promoting complex (APC), in association with its substrate-specific factor Cdh1. To dissect the potential role of Cdh1/APC in tumorigenesis through the degradation of Skp2, we analyzed the Cdh1/APC-Skp2-p27 axis in colorectal tumorigenesis using a human tumor array and biochemical analyses. Our results show that the percentage of Cdh1- and p27-positive samples in colon cancer tissues was significantly lower than that in adjacent nonmalignant tissue. Conversely, the percentage of Skp2-positive colon cancer samples was significantly higher than that in normal tissue. Furthermore, results from clinicopathological analysis revealed that elevated Cdh1 expression was associated with lower histological grade tumors. In addition, depletion of Cdh1 by RNA interference in nonmalignant colon cells resulted in increased cellular proliferation, whereas knockdown of Skp2 significantly suppressed cancer cell growth. Our result suggests a pathological correlation between Skp2 and Cdh1/APC in colorectal cancer. Thus, Cdh1 may function as a component in tumor suppression via proteolysis of Skp2 in colorectal tumorigenesis and may serve as a prognostic marker in colon cancer patients.