Epithelial-to-mesenchymal transition promotes immune escape by inducing CD70 in non-small cell lung cancer.

INTRODUCTION: Epithelial-to-mesenchymal transition (EMT) is associated with tumor aggressiveness, drug resistance, and poor survival in non-small cell lung cancer (NSCLC) and other cancers. The identification of immune-checkpoint ligands (ICPLs) associated with NSCLCs that display a mesenchymal phenotype (mNSCLC) could help to define subgroups of patients who may benefit from treatment strategies using immunotherapy. METHODS: We evaluated ICPL expression in silico in 130 NSCLC cell lines. In vitro, CRISPR/Cas9-mediated knockdown and lentiviral expression were used to assess the impact of ZEB1 expression on CD70. Gene expression profiles of lung cancer samples from the TCGA (n = 1018) and a dataset from MD Anderson Cancer Center (n = 275) were analyzed. Independent validation was performed by immunohistochemistry and targeted-RNA sequencing in 154 NSCLC whole sections, including a large cohort of pulmonary sarcomatoid carcinomas (SC, n = 55). RESULTS: We uncover that the expression of CD70, a regulatory ligand from the tumor necrosis factor ligand family, is enriched in mNSCLC in vitro models. Mechanistically, the EMT-inducer ZEB1 impacted CD70 expression and fostered increased activity of the CD70 promoter. CD70 overexpression was also evidenced in mNSCLC patient tumor samples and was particularly enriched in SC, a lung cancer subtype associated with poor prognosis. In these tumors, CD70 expression was associated with decreased CD3+ and CD8+ T-cell infiltration and increased T-cell exhaustion markers. CONCLUSION: Our results provide evidence on the pivotal roles of CD70 and ZEB1 in immune escape in mNSCLC, suggesting that EMT might promote cancer progression and metastasis by not only increasing cancer cell plasticity but also reprogramming the immune response in the local tumor microenvironment.

Somatic MMR gene mutations as a cause for MSI-H sebaceous neoplasms in Muir-Torre syndrome-like patients.

Sebaceous neoplasms are a major clinical feature of Muir-Torre syndrome (MTS) associated with visceral malignancies, especially colorectal and endometrial tumors. The diagnosis of MTS relies largely on the microsatellite instability (MSI) phenotype in tumors, suggesting germline mutations in DNA mismatch repair (MMR) genes responsible for the inherited disease. We hypothesized that in some MSI-H sebaceous tumors, acquired rather than inherited mutations in MMR genes could be involved. Using next-generation sequencing, we screened MMR gene mutations in 18 MSI-H sebaceous tumors. We found mutations in 17 samples (94%). Indeed, 12/17 (71%) were shown to carry acquired somatic mutations and among 12 samples, seven were shown to be associated with additional somatic alterations like loss of heterozygosity or multiple mutations, suggesting somatic second hits. Our findings strongly suggest that somatic MMR deficiency is responsible for a proportion of MSI-H sebaceous tumors.

Intensity-dependent constitutional MLH1 promoter methylation leads to early onset of colorectal cancer by affecting both alleles.

Constitutional epimutation is one of the causes for MLH1 gene inactivation associated with hereditary non-polyposis colon cancer (HNPCC) syndrome. Here we investigate MLH1 promoter hypermethylation in 110 sporadic early-onset colorectal cancer patients. Variable levels of hypermethylation were detected in 55 patients (50%). Importantly a reduced MLH1 gene expression was found in patients with high-level methylation, with the association of microsatellite instability (MSI) in their tumor cells. Such high-level methylation accounts for 7.4% of all patients included in this study. Furthermore, we found that in one case constitutional methylation affected both alleles, indicating a post-zygotic methylation dysregulation. Our findings suggest that constitutional epimutation is a mechanism underlying early-onset colorectal cancer, although it is involved in only a small proportion of patients, who require appropriate surveillance. Our findings provide further insight into the role of aberrant constitutional methylation in colon carcinogenesis and raise the question of whether prevalent low-level methylation constitutes a potential risk factor for cancer development.

The SNAIL family member SCRATCH1 is not expressed in human tumors.

The SNAIL and SLUG transcription factors play important roles in embryogenesis owing to their anti-apoptotic properties and their ability to promote morphogenetic changes by inducing epithelial-mesenchymal transitions (EMT). These characteristics provide many of the proteins in these families with oncogenic and pro-metastatic capabilities when reactivated in cancers. The SCRATCH subgroup of the SNAIL superfamily, including SCRATCH1 and SCRATCH2, display distinct embryonic functions and diverge early in evolution. Despite the described overexpression of SCRT1 (encoding for SCRATCH1) in a small subset of human lung cancers, there is little data supporting a role of SCRATCH proteins in tumorigenesis. To further explore this possibility, we assessed SNAI1 (SNAIL), SNAI2 (SLUG) and SCRT1 (SCRATCH1) expression in a wide panel of human and murine tumors encompassing 151 primary tumors and 6 different cancer types, including melanomas and multiple different carcinomas. Whereas SNAI1 and SNAI2 are widely expressed in human and murine tumors, our results reveal that SCRT1 transcripts are undetectable in nearly all of the examined tumors suggesting that SCRATCH1 plays a minor role, if any, in tumorigenesis. Our data therefore suggest that oncogenic properties are not shared by all SNAIL superfamily members but instead are specifically allotted to the SNAIL subgroup supporting the conclusions that SNAIL and SCRATCH subgroups are functionally divergent and strengthening the hypothesis that the oncogenic potential of SNAIL and SLUG proteins relies on the hijacking of their embryonic functions.

Weekly administration of paclitaxel induces long-term aneugenicity in nude mice.

We investigated the potential in vivo aneugenic effects associated with paclitaxel treatment. For this purpose, we treated female nude mice with paclitaxel using doses equivalent to those used in weekly schedules at the clinical level (three cycles of 30 mg/kg/week for three consecutive weeks followed by one resting week). We then evaluated the frequencies of micronucleated erythrocytes (MNE) in peripheral blood using the acridine orange micronucleus assay. The frequency of MNE was evaluated after 24 h and 168 h of administration of the last dose of each paclitaxel cycle (STA mice group) as well as after one year of the first dose of treatment (LTA mice group). We also analyzed the cytology of peripheral blood and bone marrows obtained from these mice at each time period. In the STA mice group, three cycles of paclitaxel induced a 2.4-fold increase in MNE frequencies compared to the control group (p < 0.01). This effect was observed after 24 h of the last dose of each chemotherapy cycle and persisted at least for 168 h. In the LTA mice group, paclitaxel-treated mice presented a 1.8-fold increase in the MNE frequency (p = 0.01) indicating that paclitaxel-induced MNE increase lasted for at least one year. Although the appearance of micronuclei in erythrocytes and granulocytes in peripheral blood and bone marrow cytological smears, there was no evidence of myeloproliferative disease. The present data therefore indicate an aneugenic potential of paclitaxel for humans, which should be considered in the risk-benefit analysis of its increasing clinical use.

A p21/WAF1 mutation favors the appearance of drug resistance to paclitaxel in human noncancerous epithelial mammary cells.

We investigated the mechanisms responsible for paclitaxel resistance in HME-1 cells (human mammary epithelial cells immortalized with hTERT). These cells were exposed to paclitaxel (10 pM for 7 days) and 20 cellular surviving populations (PSP) were obtained. PSP demonstrated high levels of resistance to paclitaxel cytotoxicity as compared with HME-1 cells. Activation of mdr-1 gene expression was observed in 2 PSP. Protein expression analysis using a C-terminal targeted antibody showed that 13 PSP were negative for p21/WAF1 expression after ionizing radiation (6 Gy) or doxorubicin (100 nM) treatment. Sequencing of the 3 exons of the CDKN1A gene revealed that 13 PSP contained a point mutation in exon 2. This mutation consisted in a T insertion at codon 104 leading to a premature STOP codon appearance. Mismatch amplification mutation assay and RFLP-PCR confirmed the presence of the mutation in 16 PSP. Western blot using an N-terminal targeted antibody demonstrated that the C-terminal-truncated p21/WAF1 protein (14 kDa) was indeed expressed in the 13 PSP. Our data suggest that p21/WAF1 inactivation may confer a strong resistance to paclitaxel in noncancerous breast epithelial cells harboring a p21/WAF1 mutant.

Circulating MYCN DNA as a tumor-specific marker in neuroblastoma patients.

MYCN oncogene amplification is an established indicator of the aggressiveness of neuroblastomas; it is used internationally for stratifying patients for therapy. The present study shows that high levels of MYCN DNA sequences are present in the peripheral blood of patients with MYCN-amplified neuroblastomas. Circulating MYCN DNA may be a powerful and noninvasive prognostic marker at the time of diagnosis. Furthermore, preliminary data strongly suggest that the release of MYCN sequences in the peripheral blood is an early process in disease progression, permitting us to propose this novel marker for the follow-up of patients after chemotherapy.

The human BTG2/TIS21/PC3 gene: genomic structure, transcriptional regulation and evaluation as a candidate tumor suppressor gene.

BTG2/TIS21/PC3 protein is involved in the regulation of G1/S transition of the cell cycle by inhibiting pRb function, suggesting that BTG2/TIS21/PC3 regulation is critical for normal cell growth and proliferation. To understand the regulatory mechanisms for the expression of BTG2/TIS21/PC3 we cloned the human gene. Potential binding sites for several transcription factors were identified in the 5'-flanking region of the gene. Transient expression assays with BTG2/TIS21/PC3 promoter deletions and electrophoretic mobility shift analysis identified a major wild-type p53 response element located -74 to -122 relative to the start codon. This genomic fragment was sufficient to constitute a promoter element in the presence of p53. The BTG2/TIS21/PC3 gene is an antiproliferative gene which maps within a chromosomal segment (1q32) frequently altered in breast adenocarcinomas. However, no mutations of BTG2/TIS21/PC3 were detected in breast cancer cells, suggesting that the inactivation of this gene is not a frequent genetic event during breast carcinogenesis.