Relation between outcomes and expression of estrogen receptor‐α phosphorylated at Ser167 in endometrioid endometrial cancer

Both ligand‐dependent and ligand‐independent activation of estrogen receptor (ER)α is modulated by receptor phosphorylation and results in activation of the ERα‐dependent pathways that are involved in endometrioid endometrial cancer (EEC) pathogenesis. It is also known that the mammalian target of rapamycin (mTOR)/p70 S6 kinase 1 (S6K1) and MAPK/p90 ribosomal S6 kinase (RSK) signaling pathways coordinately regulate phosphorylated‐ERα at Ser¹⁶⁷ (p‐Ser¹⁶⁷‐ERα). However, the expression of p‐Ser¹⁶⁷‐ERα in EEC and its prognostic role in ECC is largely unexplored. The purpose of the present study was to investigate the expression of p‐Ser¹⁶⁷‐ERα in ECC and its relationship with prognosis. Immunohistochemical staining of primary EEC surgical specimens (n = 103) was carried out using antibodies specific for p‐Ser¹⁶⁷‐ERα and for p‐mTOR/p‐S6K1 and p‐MAPK/p‐RSK. The correlation of p‐Ser¹⁶⁷‐ERα expression with clinicopathological features and survival of ECC was studied. Patients that were positive for nuclear p‐Ser¹⁶⁷‐ERα had significantly shorter relapse‐free survival, and although the result was not significant, levels of nuclear p‐Ser¹⁶⁷‐ERα tended to be higher in advanced‐stage ECC patients. Nuclear p‐Ser¹⁶⁷‐ERα was significantly positively correlated with p‐MAPK and p‐S6K1, and with significantly shorter relapse‐free survival in EEC.     

Regulation of peroxisome proliferator‐activated receptor‐β/δ by the APC/β‐CATENIN pathway and nonsteroidal antiinflammatory drugs

Studies indicate that peroxisome proliferator‐activated receptor‐β/δ (PPARβ/δ) can either attenuate or potentiate colon cancer. One hypothesis suggests that PPARβ/δ is upregulated by the adenomatous polyposis coli (APC)/β‐CATENIN pathway and a related hypothesis suggests that PPARβ/δ is downregulated by nonsteroidal antiinflammatory drugs (NSAIDs). The present study examined these possibilities using in vivo and in vitro models. While APC/β‐CATENIN‐dependent expression of CYCLIN D1 was observed in vivo and in vitro, expression of PPARβ/δ was not different in colon or intestinal polyps from wild‐type or Apc^min heterozygous mice or in human colon cancer cell lines with mutations in APC and/or β‐CATENIN. No difference in the level of PPARβ/δ was found in colon from wild‐type or Apc^min heterozygous mice following treatment with NO‐donating aspirin (NO‐ASA). NSAIDs inhibited cell growth in RKO (wild‐type APC) and DLD1 (mutant APC) human colon cancer cell lines but expression of PPARβ/δ was not downregulated in these cell lines in response to a broad concentration range of celecoxib, indomethacin, NS‐398, or nimesulide. However, indomethacin caused an increase in PPARβ/δ mRNA and protein that was accompanied with increased expression of a known PPARβ/δ target gene. Interestingly, expression of PPARα was also increased in the human colon cancer cell lines by several NSAIDs at the highest concentration examined. Results from these studies provide additional evidence indicating that PPARβ/δ is not upregulated by the APC/β‐CATENIN pathway. Further, these studies suggest that increased PPARβ/δ and/or PPARα by NSAIDs in human colon cancer cell lines could contribute to the mechanisms underlying the chemopreventive effects of NSAIDs. Mol. Carcinog. © 2009 Wiley‐Liss, Inc.     

Breast cancer‐derived transforming growth factor‐β and tumor necrosis factor‐α compromise interferon‐α production by tumor‐associated plasmacytoid dendritic cells

We previously reported that plasmacytoid dendritic cells (pDCs) infiltrating breast tumors are impaired for their interferon‐α (IFN‐α) production, resulting in local regulatory T cells amplification. We designed our study to decipher molecular mechanisms of such functional defect of tumor‐associated pDC (TApDC) in breast cancer. We demonstrate that besides IFN‐α, the production by Toll‐like receptor (TLR)‐activated healthy pDC of IFN‐β and TNF‐α but not IP‐10/CXCL10 nor MIP1‐α/CCL3 is impaired by the breast tumor environment. Importantly, we identified TGF‐β and TNF‐α as major soluble factors involved in TApDC functional alteration. Indeed, recombinant TGF‐β1 and TNF‐α synergistically blocked IFN‐α production of TLR‐activated pDC, and neutralization of TGF‐β and TNF‐α in tumor‐derived supernatants restored pDCs' IFN‐α production. The involvment of tumor‐derived TGF‐β was further confirmed in situ by the detection of phosphorylated Smad2 in the nuclei of TApDC in breast tumor tissues. Mechanisms of type I IFN inhibition did not involve TLR downregulation but the inhibition of IRF‐7 expression and nuclear translocation in pDC after their exposure to tumor‐derived supernatants or recombinant TGF‐β1 and TNF‐α. Our findings indicate that targeting TApDC to restore their IFN‐α production might be an achievable strategy to induce antitumor immunity in breast cancer by combining TLR7/9‐based immunotherapy with TGF‐β and TNF‐α antagonists.     

Modulation of TGF‐β activity by latent TGF‐β‐binding protein 1 in human malignant glioma cells

High biological activity of the transforming growth factor (TGF)‐β‐Smad pathway characterizes the malignant phenotype of malignant gliomas and confers poor prognosis to glioma patients. Accordingly, TGF‐β has become a novel target for the experimental treatment of these tumors. TGF‐β is processed by furin‐like proteases (FLP) and secreted from cells in a latent complex with its processed propeptide, the latency‐associated peptide (LAP). Latent TGF‐β‐binding protein 1 (LTBP‐1) covalently binds to this small latent TGF‐β complex (SLC) and regulates its function, presumably via interaction with the extracellular matrix (ECM). We report here that the levels of LTBP‐1 protein in vivo increase with the grade of malignancy in gliomas. LTBP‐1 is associated with the ECM as well as secreted into the medium in cultured malignant glioma cells. The release of LTBP‐1 into the medium is decreased by the inhibition of FLP activity. Gene‐transfer mediated overexpression of LTBP‐1 in glioma cell lines results in an increase inTGF‐β activity. Accordingly, Smad2 phosphorylation as an intracellular marker of TGF‐β activity is enhanced. Conversely, LTBP‐1 gene silencing reduces TGF‐β activity and Smad2 phosphorylation without affecting TGF‐β protein levels. Collectively, we identify LTBP‐1 as an important modulator of TGF‐β activation in glioma cells, which may contribute to the malignant phenotype of these tumors. © 2009 UICC     

Protein phosphatase 1, regulatory subunit 15B is a survival factor for ERα‐positive breast cancer

Breast cancer is a heterogeneous disease at both the clinical and molecular levels. This heterogeneity may give rise to different therapy responses. Molecular profiling has facilitated identification of signatures for stratifying patients who would potentially benefit from given therapies. Previously, we reported on a subset of genes with the potential for predicting response of primary breast cancer to neoadjuvant chemotherapy. Herein, we report that patients with luminal (estrogen receptor α [ERα]‐expressing) breast cancer were enriched for nonresponders. To identify novel factors that contribute to the survival of breast cancer cells, a loss‐of‐function screen was performed with a subset of genes overexpressed in patients with disease resistant to chemotherapy. This approach led us to identify protein phosphatase 1, regulatory subunit 15B (PPP1R15B) as a factor with a potentially essential role in the survival of ERα‐positive breast cancer cells. Functional analyses showed that PPP1R15B depletion results in impaired proliferation due to unsuccessful transition of cells from G1 to S phase of the cell cycle, and apoptosis induction. Moreover, our data revealed a regulatory role for PPP1R15B in activating ERα. Furthermore, a high level of PPP1R15B mRNA expression was associated with poor outcome following tamoxifen‐based therapy. Accordingly, knockdown of PPP1R15B expression sensitized tamoxifen‐resistant MCF‐7 breast cancer cells to tamoxifen while reducing ERα abundance in these cells. Our findings reveal a novel role for PPP1R15B in the survival and therapy response of ERα‐positive breast cancer and may open new avenues for tumor subtype‐specific therapeutic strategies in the era of personalized medicine.     

Retinoid X receptor α enhances human cholangiocarcinoma growth through simultaneous activation of Wnt/β‐catenin and nuclear factor‐κB pathways

Retinoid X receptor α (RXRα) plays important roles in the malignancy of several cancers such as human prostate tumor, breast cancer, and thyroid tumor. However, its exact functions and molecular mechanisms in cholangiocarcinoma (CCA), a chemoresistant carcinoma with poor prognosis, remain unclear. In this study we found that RXRα was frequently overexpressed in human CCA tissues and CCA cell lines. Downregulation of RXRα led to decreased expression of mitosis‐promoting factors including cyclin D1and cyclin E, and the proliferating cell nuclear antigen, as well as increased expression of cell cycle inhibitor p21, resulting in inhibition of CCA cell proliferation. Furthermore, RXRα knockdown attenuated the expression of cyclin D1 through suppression of Wnt/β‐catenin signaling. Retinoid X receptor α upregulated proliferating cell nuclear antigen expression through nuclear factor‐κB (NF‐κB) pathways, paralleled with downregulation of p21. Thus, the Wnt/β‐catenin and NF‐κB pathways account for the inhibition of CCA cell growth induced by RXRα downregulation. Retinoid X receptor α plays an important role in proliferation of CCA through simultaneous activation of Wnt/β‐catenin and NF‐κB pathways, indicating that RXRα might serve as a potential molecular target for CCA treatment.     

Transforming growth factor‐β induces CD44 cleavage that promotes migration of MDA‐MB‐435s cells through the up‐regulation of membrane type 1‐matrix metalloproteinase

CD44, a transmembrane receptor for hyaluronic acid, is implicated in various adhesion‐dependent cellular processes, including cell migration, tumor cell metastasis and invasion. Recent studies demonstrated that CD44 expressed in cancer cells can be proteolytically cleaved at the ectodomain by membrane type 1‐matrix metalloproteinase (MT1‐MMP) to form soluble CD44 and that CD44 cleavage plays a critical role in cancer cell migration. Here, we show that transforming growth factor‐β (TGF‐β), a multifunctional cytokine involved in cell proliferation, differentiation, migration and pathological processes, induces MT1‐MMP expression in MDA‐MB‐435s cells. TGF‐β‐induced MT1‐MMP expression was blocked by the specific extracellular regulated kinase‐1/2 (ERK1/2) inhibitor PD98059 and the specific phosphoinositide 3‐OH kinase (PI3K) inhibitor LY294002. In addition, treatment with SP600125, an inhibitor for c‐Jun NH₂‐terminal kinase (JNK), resulted in a significant inhibition of MT1‐MMP production. These data suggest that ERK1/2, PI3K, and JNK likely play a role in TGF‐β‐induced MT1‐MMP expression. Interestingly, treatment of MDA‐MB‐435s cells with TGF‐β resulted in a colocalization of MT1‐MMP and CD44 in the cell membrane and in an increased level of soluble CD44. Using an electric cell‐substrate impedance sensing cell‐electrode system, we demonstrated that TGF‐β treatment promotes MDA‐MB‐435s cell migration, involving MT1‐MMP‐mediated CD44 cleavage. MT1‐MMP siRNA transfection‐inhibited TGF‐β‐induced cancer cell transendothelial migration. Thus, this study contributes to our understanding of molecular mechanisms that play a critical role in tumor cell invasion and metastasis. © 2009 Wiley‐Liss, Inc.     

Opposing effects of HIF1α and HIF2α on chromaffin cell phenotypic features and tumor cell proliferation: Insights from MYC‐associated factor X

Pheochromocytomas and paragangliomas (PPGLs) are catecholamine‐producing chromaffin cell tumors with diverse phenotypic features reflecting mutations in numerous genes, including MYC‐associated factor X (MAX). To explore whether phenotypic differences among PPGLs reflect a MAX‐mediated mechanism and opposing influences of hypoxia‐inducible factor (HIF)s HIF2α and HIF1α, we combined observational investigations in PPGLs and gene‐manipulation studies in two pheochromocytoma cell lines. Among PPGLs from 140 patients, tumors due to MAX mutations were characterized by gene expression profiles and intermediate phenotypic features that distinguished these tumors from other PPGLs, all of which fell into two expression clusters: one cluster with low expression of HIF2α and mature phenotypic features and the other with high expression of HIF2α and immature phenotypic features due to mutations stabilizing HIFs. Max‐mutated tumors distributed to a distinct subcluster of the former group. In cell lines lacking Max, re‐expression of the gene resulted in maturation of phenotypic features and decreased cell cycle progression. In cell lines lacking Hif2α, overexpression of the gene led to immature phenotypic features, failure of dexamethasone to induce differentiation and increased proliferation. HIF1α had opposing actions to HIF2α in both cell lines, supporting evolving evidence of their differential actions on tumorigenic processes via a MYC/MAX‐related pathway. Requirement of a fully functional MYC/MAX complex to facilitate differentiation explains the intermediate phenotypic features in tumors due to MAX mutations. Overexpression of HIF2α in chromaffin cell tumors due to mutations affecting HIF stabilization explains their proliferative features and why the tumors fail to differentiate even when exposed locally to adrenal steroids.     

Effects of α‐tocopherol and β‐carotene supplementation on cancer incidence and mortality: 18‐Year postintervention follow‐up of the Alpha‐Tocopherol,Beta‐Carotene Cancer Prevention Study

In the Alpha‐Tocopherol, Beta‐Carotene Cancer Prevention Study among 29,133 Finnish male smokers aged 50–69 years, daily α‐tocopherol (50 mg) for a median of 6.1 years decreased the risk of prostate cancer, whereas β‐carotene (20 mg) increased risk of lung cancer and overall mortality. To determine the postintervention effects of α‐tocopherol and β‐carotene, 25,563 men were followed 18 years for cancer incidence and all causes of mortality through national registers. Neither supplement had significant effects on post‐trial cancer incidence. Relative risk (RR) for lung cancer (n = 2,881) was 1.04 (95% confidence interval [CI], 0.96–1.11) among β‐carotene recipients compared with nonrecipients. For prostate cancer (n = 2,321), RR was 0.97 (95% CI, 0.89–1.05) among α‐tocopherol recipients compared with nonrecipients with the preventive effect of α‐tocopherol continuing ～8 years postintervention. Body mass index significantly modified the effect of α‐tocopherol on prostate cancer (p for interaction = 0.01) RR 1.00 (95% CI, 0.88–1.14) in normal‐weight men, 0.87 (95% CI, 0.77–0.98) in overweight men, and 1.25 (95% CI, 1.01–1.55) in obese men. The post‐trial relative mortality (based on 16,686 deaths) was 1.02 (95% CI, 0.98–1.05) for α‐tocopherol recipients compared with nonrecipients and 1.02 (95% CI, 0.99–1.05) for β‐carotene recipients compared with nonrecipients. α‐Tocopherol decreased post‐trial prostate cancer mortality (RR, 0.84; 95% CI, 0.70–0.99), whereas β‐carotene increased it (RR, 1.20; 95% CI, 1.01–1.42). In conclusion, supplementation with α‐tocopherol and β‐carotene appeared to have no late effects on cancer incidence. The preventive effect of moderate‐dose α‐tocopherol on prostate cancer continued several years post‐trial and resulted in lower prostate cancer mortality.     

Estrogen receptor‐β gene polymorphism and colorectal cancer risk: Effect modified by body mass index and isoflavone intake

Estrogen receptor (ER)‐β signaling has generally been implicated in protection against colorectal cancer. The ER‐β gene cytosine‐adenine (ESR2 CA) repeat polymorphism was reported to be associated with colorectal cancer, although showing contradicting results probably caused by ethnicity or age distribution of the subjects. We investigated the association between this polymorphism and the colorectal cancer risk in a community‐based case‐control study in Japan (685 cases/778 controls), including only subjects younger than 75. The effect modifications of the body mass index (BMI) and isoflavone intake were also examined. ESR2 CA repeat polymorphism was determined by polymerase chain reaction using fluorescein‐labeled primers. CA repeat alleles were classified into short (S) allele (<22 repeats) and long (L) allele (≥22 repeats). Subjects were divided into three genotype groups (SS/SL/LL). The risk of colon cancer, but not of rectal cancer, was increased with an increasing number of L alleles among postmenopausal women; age‐adjusted odds ratio (OR) for SL and LL genotypes compared with the SS genotype were 1.78 and 2.91, respectively (trend p = 0.002). Increased risks of colon cancer associated with the L allele were more evident among postmenopausal women with low BMI (<25 kg m^?2) or with high isoflavone intake. Such associations were not observed among men or premenopausal women. Having longer ESR2 CA repeat increases colon cancer risk among postmenopausal women younger than 75, possibly with modification of BMI and isoflavone intake. Aging and estrogenic condition may be important in the colon cancer pathogenesis associated with ESR2 CA repeat polymorphism.     

Clinicopathological analysis of 17 primary cutaneous T‐cell lymphoma of the γδ phenotype from Japan

Primary cutaneous γδ T‐cell lymphoma (PCGD‐TCL) is an aggressive lymphoma consisting of clonal proliferation of mature activated γδ T‐cells of a cytotoxic phenotype. Because primary cutaneous γδ T‐cell lymphoma is a rare disease, there are few clinicopathological studies. In addition, T‐cell receptor (TCR) γδ cells are typically immunostained in frozen sections or determined by TCRβ negativity. We retrospectively analyzed 17 primary cutaneous T‐cell lymphomas of the γδ phenotype (CTCL‐γδ) in a clinicopathological and molecular study using paraffin‐embedded sections. Among 17 patients, 11 had CTCL‐γδ without subcutaneous panniculitis‐like T‐cell lymphoma (SPTCL) features and six had CTCL‐γδ with SPTCL features. Immunophenotypically, some significant differences were found in CD8 and CD56 positivity between our patient series of CTCL‐γδ patients with SPTCL features and SPTCL‐γδ patients described in the previous literature. A univariate analysis of 17 CTCL‐γδ patients showed that being more than 60 years old, presence of visceral organ involvement, and small‐to‐medium cell size were poor prognostic factors. In addition, the 5‐year overall survival rate was 42.4% for the CTCL‐γδ patients without SPTCL features and 80.0% for those with SPTCL features. Consequently, there was a strikingly significant difference in overall survival among SPTCL, CTCL‐γδ with SPTCL features and CTCL‐γδ without SPTCL features (P = 0.0005). Our data suggests that an indolent subgroup may exist in CTCL‐γδ. Studies on more cases, including those from other countries, are warranted to delineate the clinicopathological features and the significance in these rare lymphomas.     

Endometrial cancer invasion depends on cancer‐derived tumor necrosis factor‐α and stromal derived hepatocyte growth factor

Cancer invasion is an outcome of interactions of the cancer and the host cell. It is now becoming increasingly clear that ovarian hormones have a huge influence on such intercommunications in various types of cancers. Estrogen is known to aggravate the aggressiveness of the endometrial cancer whereas progesterone seems to act as a negative factor. Insight into the mode of ovarian hormonal actions could come from the studies of its regulation of the paracrine interactions between the endometrial cancer and the normal stromal cells during the cancer invasion. In this context, we report here that estrogen promotes the endometrial cancer invasion by inducing humoral interactions between the cancer and the stromal cells, i.e., estrogen stimulates tumor necrosis factor‐α expression from the endometrial cancer cells, which, in turn, induces the stromal expression of hepatocyte growth factor (HGF), conferring the enhanced NK4 (HGF‐antagonist/angiogenesis inhibitor)‐sensitive invasion characteristic of the endometrial cancer cells. Additionally, we demonstrate a close correlation of the invasion of endometrial cancer cells with the expression and dimerization of integrin α_vβ₅ as well as the activation of focal adhesion kinase as the consequences of paracrine interactions. Thus, understanding of paracrine interactions of cancer cells with host stromal cells can yield new insight into the architecture and function of cancer invasion and metastasis, leading to a development of a new cancer therapeutic intervention. © 2008 Wiley‐Liss, Inc.     

Bisphosphonates suppress insulin‐like growth factor 1‐induced angiogenesis via the HIF‐1α/VEGF signaling pathways in human breast cancer cells

Adjunctive chemotherapy with bisphosphonates has been reported to delay bone metastasis and improve overall survival in breast cancer. Aside from its antiresorptive effect, bisphosphonates exhibit antitumor activities, in vitro and in vivo, via several mechanisms, including antiangiogenesis. In this study, we investigated the potential molecular mechanisms underlying the antiangiogenic effect of non–nitrogen‐containing and nitrogen‐containing bisphosphonates, clodronate and pamidronate, respectively, in insulin‐like growth factor (IGF)‐1 responsive human breast cancer cells. We tested whether bisphosphonates had any effects on hypoxia‐inducible factor (HIF)‐1α/vascular endothelial growth factor (VEGF) axis that plays a pivotal role in tumor angiogenesis, and our results showed that both pamidronate and clodronate significantly suppressed IGF‐1‐induced HIF‐1α protein accumulation and VEGF expression in MCF‐7 cells. Mechanistically, we found that either pamidronate or clodronate did not affect mRNA expression of HIF‐1α, but they apparently promoted the degradation of IGF‐1‐induced HIF‐1α protein. Meanwhile, we found that the presence of pamidronate and clodronate led to a dose‐dependent decease in the newly‐synthesized HIF‐1α protein induced by IGF‐1 in breast cancer cells after proteasomal inhibition, thus, indirectly reflecting the inhibition of protein synthesis. In addition, our results indicated that the inhibitory effects of bisphosphonates on the HIF‐1α/VEGF axis are associated with the inhibition of the phosphoinositide 3‐kinase/AKT/mammalian target of rapamycin signaling pathways. Consistently, we demonstrated that pamidronate and clodronate functionally abrogated both in vitro and in vivo tumor angiogenesis induced by IGF‐1‐stimulated MCF‐7 cells. These findings have highlighted an important mechanism of the pharmacological action of bisphosphonates in the inhibition of tumor angiogenesis in breast cancer cells.