Determination of the Expression of PD-L1 in the Morphologic Spectrum of Renal Cell Carcinoma.

Immunotherapy is reportedly an effective form of therapy for some advanced cancers such as lung adenocarcinoma, malignant melanoma and colorectal adenocarcinoma. In renal cell carcinoma (RCC), the role of immunotherapy is under investigation. Programmed Death-Ligand 1 (PD-L1) is a molecule expressed on the surface of certain tumor cells and binds to the Programmed cell death protein 1 (PD-1) on cytotoxic T-cells, an interaction that inhibits the antitumor immune response. The aim of this study is to evaluate PD-L1 expression in the morphologic spectrum of RCC. A total of 172 cases of RCC comprising all types were studied and the PD-L1 was correlated with immune response for CD4 and CD8. Positive membranous staining for PD-L1 was seen in 59 (34%) of the 172 samples. The positive cases were HLRCC (31/53), Type 1 Papillary RCC (10/31), Chromophobe (7/20), Hybrid (3/9), TFE-3 related cancer (3/8), Undifferentiated (3/5), and TFEB tumors (2/2). Clear cell carcinomas, Oncocytomas and SDHB deficient-RCC didn't show any expression of PD-L1; (0/34;0/7;0/3). Our results demonstrated that aggressive forms of RCC such as HLRCC have high expression of PD-L1, in contrast to clear cell renal carcinomas. Our findings support a possible role of anti-PD-L1/PD-1 immunotherapies in the treatment of PD-L1-positive RCC.

Spatiotemporal Expression of Three Secretoglobin Proteins, SCGB1A1, SCGB3A1, and SCGB3A2, in Mouse Airway Epithelia.

Secretoglobins (SCGBs) are cytokine-like small molecular weight secreted proteins with largely unknown biological functions. Three SCGB proteins, SCGB1A1, SCGB3A1, and SCGB3A2, are predominantly expressed in lung airways. To gain insight into the possible functional relationships among the SCGBs, their protein and mRNA expression patterns were examined in lungs during gestation and in adult mice, using Scgb3a1-null and Scgb3a2-null mice as negative controls, by immunohistochemistry and by qRT-PCR analysis, respectively. The three SCGBs exhibited unique spatiotemporal expression patterns during embryogenesis. The lack of Scgb3a1 or Scgb3a2 did not affect expression of the other Scgb genes as determined by mRNA measurements. Moreover, the lack of Scgb3a1 or Scgb3a2 did not affect development of the pulmonary neuroepithelial bodies during embryogenesis, while the lack of Scgb3a2 may have resulted in slightly fewer ciliated cells than in the wild-type. These results suggest that SCGB1A1, SCGB3A1, and SCGB3A2 each may possess its own unique biological function.

Co-expression of Achaete-Scute Homologue-1 and Calcitonin Gene-Related Peptide during NNK-Induced Pulmonary Neuroendocrine Hyperplasia and Carcinogenesis in Hamsters.

Achaete-scute homologue-1 or ASCL1 (MASH1, hASH1) plays roles in neural development and pulmonary neuroendocrine (NE) differentiation, and it is expressed in certain lung cancers. This study was aimed to assess whether and/or how ASCL1 plays a role in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced pulmonary NE hyperplasia and carcinogenesis in hamsters. Hamsters were injected 3 times weekly with either NNK or solvent alone (control) for treatment periods of 6 and 24 weeks, both without and with 6-week recovery. Immunohistochemical analysis was carried out to examine the expressions of ASCL1, CGRP (calcitonin gene-related peptide), secretoglobin SCGB1A1 (club [Clara] cell specific 10 kD protein, CC10, CCSP), synaptophysin (SYP), and PCNA (proliferating cell nuclear antigen). The number of ASCL1-expressing NE foci per airway increased from 0.8 in controls to 1.6 and 2.0 during NNK exposure for 6 and 24 weeks, respectively, and the number of cells per foci doubled after NNK exposure. Most ASCL1-expressing cells in NEBs (neuroepithelial bodies) were also CGRP immunoreactive; NNK enhanced this co-expression with CGRP, a NE marker with known proliferation-promoting properties. NNK also increased PCNA expression within NE foci. NNK-induced tumors showed no immunoreactivity for NE markers. This study confirms ASCL1 as an excellent marker for pulmonary NE cells and demonstrates CGRP co-expression in ASCL1-positive NEB cells participating in NNK-induced NE hyperplasia.

Achaete-scute homologue-1 (ASH1) stimulates migration of lung cancer cells through Cdk5/p35 pathway.

Our previous data suggested that the human basic helix-loop-helix transcription factor achaete-scute homologue-1 (hASH1) may stimulate both proliferation and migration in the lung. In the CNS, cyclin-dependent kinase 5 (Cdk5) and its activator p35 are important for neuronal migration that is regulated by basic helix-loop-helix transcription factors. Cdk5/p35 may also play a role in carcinogenesis. In this study, we found that the neuronal activator p35 was commonly expressed in primary human lung cancers. Cdk5 and p35 were also expressed by several human lung cancer cell lines and coupled with migration and invasion. When the kinase activity was inhibited by the Cdk5 inhibitor roscovitine or dominant-negative (dn) Cdk5, the migration of lung cancer cells was reduced. In neuroendocrine cells expressing hASH1, such as a pulmonary carcinoid cell line, knocking down the gene expression by short hairpin RNA reduced the levels of Cdk5/p35, nuclear p35 protein, and migration. Furthermore, expression of hASH1 in lung adenocarcinoma cells normally lacking hASH1 increased p35/Cdk5 activity and enhanced cellular migration. We were also able to show that p35 was a direct target for hASH1. In conclusion, induction of Cdk5 activity is a novel mechanism through which hASH1 may regulate migration in lung carcinogenesis.

Secretoglobin 3A2/uteroglobin-related protein 1 is a novel marker for pulmonary carcinoma in mice and humans.

Secretoglobin (SCGB) 3A2, also called uteroglobin-related protein (UGRP) 1, is a downstream target for a homeodomain transcription factor NKX2-1, which is critical for the development of lung, thyroid and ventral forebrain. Both SCGB3A2 and NKX2-1 are expressed in airway epithelial cells and the latter also in alveolar Type II cells. NKX2-1 has been used clinically for diagnosis of human pulmonary tumors. Recently, the expression of SCGB3A2 was reported in human carcinomas, suggesting the use of this protein as a tumor marker. In this study, 28 lung tumors from aging B6;129 mice and nine lung adenocarcinomas from CC10TAg transgenic mice that express SV40 large T antigen under the mouse Scgb1a1 (CC10) gene promoter, were subjected to histopathological and immunohistochemical analyses for the expression of NKX2-1 and SCGB3A2. NKX2-1 was expressed in all types of tumors albeit more focally in carcinomas. In contrast, SCGB3A2 normally expressed in Clara cells, was negative in Type II cell hyperplasias and adenomas. However, it was expressed in alveolar Type II cell carcinomas and Clara cell adenocarcinomas. In these carcinomas, SCGB3A2 expression was observed in the portion of the tumor where NKX2-1 expression was reduced or almost abolished. As a comparison, the expression of SCGB3A2 and NKX2-1 from 23 human non-small cell lung carcinoma specimens was also examined. The results demonstrate that SCGB3A2 is a useful marker for diagnosis of pulmonary tumors both in mice and humans.

Acute damage by naphthalene triggers expression of the neuroendocrine marker PGP9.5 in airway epithelial cells.

Protein Gene Product 9.5 (PGP9.5) is highly expressed in nervous tissue. Recently PGP9.5 expression has been found to be upregulated in the pulmonary epithelium of smokers and in non-small cell lung cancer, suggesting that it also plays a role in carcinogen-inflicted lung epithelial injury and carcinogenesis. We investigated the expression of PGP9.5 in mice in response to two prominent carcinogens found in tobacco smoke: Naphthalene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). By immunostaining, we found that PGP9.5 protein was highly expressed throughout the airway epithelium in the days immediately following a single injection of naphthalene. In contrast, PGP9.5 was exclusively confined to neurons and neuroendocrine cells in the control and NNK-exposed lungs. Furthermore, we investigated the expression of PGP9.5 mRNA in the lungs by quantitative RT-PCR (qPCR). PGP9.5 mRNA expression was highly upregulated in the days immediately following naphthalene injection and gradually returning to that of control mice 5 days after naphthalene injection. In contrast, exposure to NNK did not result in a significant increase in PGP9.5 mRNA 10 weeks after exposure. No increased expression of two other neuroendocrine markers was found in the non-neuroendocrine epithelial cells after naphthalene exposure. In contrast, immunostaining for the cell cycle regulator p27(Kip1), which has previously been associated with PGP9.5 in lung cancer cells, revealed transient downregulation of p27(Kip1) in naphthalene exposed airways compared to controls, indicating that the rise in PGP9.5 in the airway epithelium is related to downregulation of p27(Kip1). This study is the first to specifically identify the carcinogen naphthalene as an inducer of PGP9.5 expression in non-neuroendocrine epithelium after acute lung injury and further strengthens the accumulating evidence of PGP9.5 as a central player in lung epithelial damage and early carcinogenesis.

Achaete-scute homolog-1 linked to remodeling and preneoplasia of pulmonary epithelium.

The basic helix-loop-helix protein achaete-scute homolog-1 (ASH1) is involved in lung neuroendocrine (NE) differentiation and tumor promotion in SV40 transgenic mice. Constitutive expression of human ASH-1 (hASH1) in mouse lung results in hyperplasia and remodeling that mimics bronchiolization of alveoli (BOA), a potentially premalignant lesion of human lung carcinomas. We now show that this is due to sustained cellular proliferation in terminal bronchioles and resistance to apoptosis. Throughout the airway epithelium the expression of anti-apoptotic Bcl-2 and c-Myb was increased and Akt/mTOR pathway activated. Moreover, the expression of matrix metalloproteases (MMPs) including MMP7 was specifically enhanced at the bronchiolo-alveolar duct junction and BOA suggesting that MMPs play a key role in this microenvironment during remodeling. We also detected MMP7 in 70% of human BOA lesions. Knockdown of hASH1 gene in human lung cancer cells in vitro suppressed growth by increasing apoptosis. We also show that forced expression of hASH1 in immortalized human bronchial epithelial cells decreases apoptosis. We conclude that the impact of hASH1 is not limited to cells with NE phenotype. Rather, constitutive expression of hASH1 in lung epithelium promotes remodeling through multiple pathways that are commonly activated during lung carcinogenesis. The collective results suggest a novel model of BOA formation via hASH1-induced suppression of the apoptotic pathway. Our study yields a promising new preclinical tool for chemoprevention of peripheral lung carcinomas.

Mouse lung neuroendocrine carcinomas: distinct morphologies, same transcription factors.

Constitutive expression of human achaete-scute homolog-1 (hASH-1) in combination with simian virus large Tantigen under the Clara cell 10-kDa secretory protein (CC10) promoter results in adenocarcinomas with focal neuroendocrine (NE) differentiation. Mice carrying conditional alleles for both Rb-1 and p53 in lung epithelial cells develop aggressive lung tumors with similarities to human small cell lung cancers, including high level expression of ASH-1, NE markers, and extra-pulmonary metastases. Tumors in both models originate from bronchiolar epithelium, reveal a range of premalignant changes, express thyroid transcription factor-1 (TTF-1), a marker of peripheral airway cell lineage, and display varying degrees of bidirectional epithelial/NE differentiation.