Regulatory mechanisms mediated by peroxisome proliferator‐activated receptor‐β/δ in skin cancer

Considerable progress has been made during the past 20 years towards elucidating the role of peroxisome proliferator‐activated receptor‐β/δ (PPARβ/δ) in skin cancer. In 1999, the original notion that PPARβ/δ was involved with epithelial cell function was postulated based on a correlation between PPARβ/δ expression and the induction of messenger RNAs encoding proteins that mediate terminal differentiation in keratinocytes. Subsequent studies definitively revealed that PPARβ/δ could induce terminal differentiation and inhibit proliferation of keratinocytes. Molecular mechanisms have since been discovered to explain how this nuclear receptor can be targeted for preventing and treating skin cancer. This includes the regulation of terminal differentiation, mitotic signaling, endoplasmic reticulum stress, and cellular senescence. Interestingly, the effects of activating PPARβ/δ can preferentially target keratinocytes with genetic mutations associated with skin cancer. This review provides the history and current understanding of how PPARβ/δ can be targeted for both nonmelanoma skin cancer and melanoma and postulates how future approaches that modulate PPARβ/δ signaling may be developed for the prevention and treatment of these diseases.     

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.     

TGF‐β inactivation and TGF‐α overexpression cooperate in an in vivo mouse model to induce hepatocellular carcinoma that recapitulates molecular features of human liver cancer

Hepatocellular carcinoma (HCC) results from the cumulative effects of deregulated tumor suppressor genes and oncogenes. The tumor suppressor and oncogenes commonly affected include growth factors, receptors and their downstream signaling pathway components. The overexpression of transforming growth factor alpha (TGF‐α) and the inhibition of TGF‐β signaling are especially common in human liver cancer. Thus, we assessed whether TGF‐α overexpression and TGF‐β signaling inactivation cooperate in hepatocarcinogenesis using an in vivo mouse model, MT1/TGFa;AlbCre/Tgfbr2^flx/flx mice (“TGFa;Tgfbr2^hepko”), which overexpresses TGF‐α and lacks a TGF‐β receptor in the liver. TGF‐β signaling inactivation did not alter the frequency or number of cancers in mice with overexpression of TGF‐α. However, the tumors in the TGFa;Tgfbr2^hepko mice displayed increased proliferation and increased cdk2, cyclin E and cyclin A expression as well as decreased Cdkn1a/p21 expression compared to normal liver and compared to the cancers arising in the TGF‐α overexpressing mice with intact TGF‐β receptors. Increased phosphorylated ERK1/2 expression was also present in the tumors from the TGFa;Tgfbr2^hepko mice and correlated with downregulated Raf kinase inhibitor protein expression, which is a common molecular event in human HCC. Thus, TGF‐β signaling inactivation appears to cooperate with TGF‐α in vivo to promote the formation of liver cancer that recapitulates molecular features of human HCC.     

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.     

TNF‐α enhances TGF‐β‐induced endothelial‐to‐mesenchymal transition via TGF‐β signal augmentation

The tumor microenvironment (TME) consists of various components including cancer cells, tumor vessels, cancer‐associated fibroblasts (CAFs), and inflammatory cells. These components interact with each other via various cytokines, which often induce tumor progression. Thus, a greater understanding of TME networks is crucial for the development of novel cancer therapies. Many cancer types express high levels of TGF‐β, which induces endothelial‐to‐mesenchymal transition (EndMT), leading to formation of CAFs. Although we previously reported that CAFs derived from EndMT promoted tumor formation, the molecular mechanisms underlying these interactions remain to be elucidated. Furthermore, tumor‐infiltrating inflammatory cells secrete various cytokines, including TNF‐α. However, the role of TNF‐α in TGF‐β‐induced EndMT has not been fully elucidated. Therefore, this study examined the effect of TNF‐α on TGF‐β‐induced EndMT in human endothelial cells (ECs). Various types of human ECs underwent EndMT in response to TGF‐β and TNF‐α, which was accompanied by increased and decreased expression of mesenchymal cell and EC markers, respectively. In addition, treatment of ECs with TGF‐β and TNF‐α exhibited sustained activation of Smad2/3 signals, which was presumably induced by elevated expression of TGF‐β type I receptor, TGF‐β2, activin A, and integrin αv, suggesting that TNF‐α enhanced TGF‐β‐induced EndMT by augmenting TGF‐β family signals. Furthermore, oral squamous cell carcinoma‐derived cells underwent epithelial‐to‐mesenchymal transition (EMT) in response to humoral factors produced by TGF‐β and TNF‐α‐cultured ECs. This EndMT‐driven EMT was blocked by inhibiting the action of TGF‐βs. Collectively, our findings suggest that TNF‐α enhances TGF‐β‐dependent EndMT, which contributes to tumor progression.     

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     

IL‐17A‐producing CD30+ Vδ1 T cells drive inflammation‐induced cancer progression

Although it has been suspected that inflammation is associated with increased tumor metastasis, the exact type of immune response required to initiate cancer progression and metastasis remains unknown. In this study, by using an in vivo tumor progression model in which low tumorigenic cancer cells acquire malignant metastatic phenotype after exposure to inflammation, we found that IL‐17A is a critical cue for escalating cancer cell malignancy. We further demonstrated that the length of exposure to an inflammatory microenvironment could be associated with acquiring greater tumorigenicity and that IL‐17A was critical for amplifying such local inflammation, as observed in the production of IL‐1β and neutrophil infiltration following the cross‐talk between cancer and host stromal cells. We further determined that γδT cells expressing Vδ1 semi‐invariant TCR initiate cancer‐promoting inflammation by producing IL‐17A in an MyD88/IL‐23‐dependent manner. Finally, we identified CD30 as a key molecule in the inflammatory function of Vδ1T cells and the blockade of this pathway targeted this cancer immune‐escalation process. Collectively, these results reveal the importance of IL‐17A‐producing CD30⁺ Vδ1T cells in triggering inflammation and orchestrating a microenvironment leading to cancer progression.     

PD‐L1 expression enhancement by infiltrating macrophage‐derived tumor necrosis factor‐α leads to poor pancreatic cancer prognosis

Immunotherapy using anti‐PD‐1/PD‐L1 antibodies for several types of cancer has received considerable attention in recent decades. However, the molecular mechanism underlying PD‐L1 expression in pancreatic ductal adenocarcinoma (PDAC) cells has not been clearly elucidated. We investigated the clinical significance and regulatory mechanism of PD‐L1 expression in PDAC cells. Among the various cytokines tested, tumor necrosis factor (TNF)‐α upregulated PD‐L1 expression in PDAC cells through NF‐κB signaling. The induction of PD‐L1 expression was also caused by co‐culture with activated macrophages, and the upregulation was inhibited by neutralization with anti‐TNF‐α antibody after co‐culture with activated macrophages. PD‐L1 expression in PDAC cells was positively correlated with macrophage infiltration in tumor stroma of human PDAC tissues. In addition, survival analysis revealed that high PD‐L1 expression was significantly associated with poor prognosis in 235 PDAC patients and especially in patients harboring high CD8‐positive T‐cell infiltration. These findings indicate that tumor‐infiltrating macrophage‐derived TNF‐α could be a potential therapeutic target for PDAC.     

CXCR2‐mediated tumor‐associated neutrophil recruitment is regulated by IFN‐β

The chemokine receptor CXCR2 and its ligands CXCL1, CXCL2 and CXCL5 play an important role in homing of tumor‐associated neutrophils (TANs) into developing tumors. TANs are known to support the development of blood vessels in growing solid tumors, hence contributing to tumor growth. Here, we show that the migration of neutrophils is influenced by endogenous interferon‐beta (IFN‐β) via regulation of such chemokines and their receptor. We could demonstrate that CXCL1 and CXCL2 gradients are formed in tumor‐bearing mice, i.e., low chemokine level in bone marrow (BM) and high level in the tumor. This supports migration of neutrophils into the tumor. Moreover, expression of CXCR2 was highest on neutrophils from BM and lowest in TANs. Importantly, although IFN‐β appears to have only a minor influence on the expression of CXCR2, it strongly regulates the CXCR2 ligands. In the absence of endogenous IFN‐β, they were expressed significantly higher in tumor‐infiltrating neutrophils. Treatment of such neutrophils from tumor‐bearing Ifnb1^?/? mice with recombinant IFN‐β downregulated CXCR2 ligand expression to wild‐type levels. This explains the reduced migration of neutrophils into tumors and the diminished tumor angiogenesis in IFN‐β‐sufficient mice. Our results add a novel functional aspect of the type I IFN system as effector molecules of natural cancer surveillance and open interesting possibilities for antineutrophil therapies against cancer.     

Repressive role of stabilized hypoxia inducible factor 1α expression on transforming growth factor β‐induced extracellular matrix production in lung cancer cells

Activation of transforming growth factor β (TGF‐β) combined with persistent hypoxia often affects the tumor microenvironment. Disruption of cadherin/catenin complexes induced by these stimulations yields aberrant extracellular matrix (ECM) production, characteristics of epithelial‐mesenchymal transition (EMT). Hypoxia‐inducible factors (HIF), the hallmark of the response to hypoxia, play differential roles during development of diseases. Recent studies show that localization of cadherin/catenin complexes at the cell membrane might be tightly regulated by protein phosphatase activity. We aimed to investigate the role of stabilized HIF‐1α expression by protein phosphatase activity on dissociation of the E‐cadherin/β‐catenin complex and aberrant ECM expression in lung cancer cells under stimulation by TGF‐β. By using lung cancer cells treated with HIF‐1α stabilizers or carrying doxycycline‐dependent HIF‐1α deletion or point mutants, we investigated the role of stabilized HIF‐1α expression on TGF‐β‐induced EMT in lung cancer cells. Furthermore, the underlying mechanisms were determined by inhibition of protein phosphatase activity. Persistent stimulation by TGF‐β and hypoxia induced EMT phenotypes in H358 cells in which stabilized HIF‐1α expression was inhibited. Stabilized HIF‐1α protein expression inhibited the TGF‐β‐stimulated appearance of EMT phenotypes across cell types and species, independent of de novo vascular endothelial growth factor A (VEGFA) expression. Inhibition of protein phosphatase 2A activity abrogated the HIF‐1α‐induced repression of the TGF‐β‐stimulated appearance of EMT phenotypes. This is the first study to show a direct role of stabilized HIF‐1α expression on inhibition of TGF‐β‐induced EMT phenotypes in lung cancer cells, in part, through protein phosphatase activity.     

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.     

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.