The immune microenvironment of HPV-negative oral squamous cell carcinoma from never-smokers and never-drinkers patients suggests higher clinical benefit of IDO1 and PD1/PD-L1 blockade.

BACKGROUND: Never-smokers and never-drinkers patients (NSND) suffering from oral squamous cell carcinoma (OSCC) are epidemiologically different from smokers drinkers (SD). We therefore hypothesized that they harbored distinct targetable molecular alterations. PATIENTS AND METHODS: Data from The Cancer Genome Atlas (TCGA) (discovery set), Gene Expression Omnibus and Centre Léon Bérard (CLB) (three validation sets) with available gene expression profiles of HPV-negative OSCC from NSND and SD were mined. Protein expression profiles and genomic alterations were also analyzed from TCGA, and a functional pathway enrichment analysis was carried out. Formalin-fixed paraffin-embedded samples from 44 OSCC including 20 NSND and 24 SD treated at CLB were retrospectively collected to perform targeted-sequencing of 2559 transcripts (HTG EdgeSeq system), and CD3, CD4, CD8, IDO1, and PD-L1 expression analyses by immunohistochemistry (IHC). Enrichment of a six-gene interferon-γ signature of clinical response to pembrozulimab (PD-1 inhibitor) was evaluated in each sample from all cohorts, using the single sample gene set enrichment analysis method. RESULTS: A total of 854 genes and 29 proteins were found to be differentially expressed between NSND and SD in TCGA. Functional pathway analysis highlighted an overall enrichment for immune-related pathways in OSCC from NSND, especially involving T-cell activation. Interferon-γ response and PD1 signaling were strongly enriched in NSND. IDO1 and PD-L1 were overexpressed and the score of response to pembrolizumab was higher in NSND than in SD, although the mutational load was lower in NSND. IHC analyses in the CLB cohort evidenced IDO1 and PD-L1 overexpression in tumor cells that was associated with a higher rate of tumor-infiltrating T-cells in NSND compared with SD. CONCLUSION: The main biological and actionable difference between OSCC from NSND and SD lies in the immune microenvironment, suggesting a higher clinical benefit of PD-L1 and IDO1 inhibition in OSCC from NSND.

Identification of BTG2, an antiproliferative p53-dependent component of the DNA damage cellular response pathway.

Cell cycle regulation is critical for maintenance of genome integrity. A prominent factor that guarantees genomic stability of cells is p53 (ref. 1). The P53 gene encodes a transcription factor that has a role as a tumour suppressor. Identification of p53-target genes should provide greater insight into the molecular mechanisms that mediate the tumour suppressor activities of p53. The rodent Pc3/Tis21 gene was initially described as an immediate early gene induced by tumour promoters and growth factors in PC12 and Swiss 3T3 cells. It is expressed in a variety of cell and tissue types and encodes a remarkably labile protein. Pc3/Tis21 has a strong sequence similarity to the human antiproliferative BTG1 gene cloned from a chromosomal translocation of a B-cell chronic lymphocytic leukaemia. This similarity led us to speculate that BTG1 and the putative human homologue of Pc3/Tis21 (named BTG2) were members of a new family of genes involved in growth control and/or differentiation. This hypothesis was recently strengthened by the identification of a new antiproliferative protein, named TOB, which shares sequence similarity with BTG1 and PC3/TIS21 (ref. 7). Here, we cloned and localized the human BTG2 gene. We show that BTG2 expression is induced through a p53-dependent mechanism and that BTG2 function may be relevant to cell cycle control and cellular response to DNA damage.

Prognostic value of Dicer expression in human breast cancers and association with the mesenchymal phenotype.

BACKGROUND: Dicer, a ribonuclease, is the key enzyme required for the biogenesis of microRNAs and small interfering RNAs and is essential for both mammalian development and cell differentiation. Recent evidence indicates that Dicer may also be involved in tumourigenesis. However, no studies have examined the clinical significance of Dicer at both the RNA and the protein levels in breast cancer. METHODS: In this study, the biological and prognostic value of Dicer expression was assessed in breast cancer cell lines, breast cancer progression cellular models, and in two well-characterised sets of breast carcinoma samples obtained from patients with long-term follow-up using tissue microarrays and quantitative reverse transcription-PCR. RESULTS: We have found that Dicer protein expression is significantly associated with hormone receptor status and cancer subtype in breast tumours (ER P=0.008; PR P=0.019; cancer subtype P=0.023, luminal A P=0.0174). Dicer mRNA expression appeared to have an independent prognostic impact in metastatic disease (hazard ratio=3.36, P=0.0032). In the breast cancer cell lines, lower Dicer expression was found in cells harbouring a mesenchymal phenotype and in metastatic bone derivatives of a breast cancer cell line. These findings suggest that the downregulation of Dicer expression may be related to the metastatic spread of tumours. CONCLUSION: Assessment of Dicer expression may facilitate prediction of distant metastases for patients suffering from breast cancer.

Properties of the six isoforms of p63: p53-like regulation in response to genotoxic stress and cross talk with DeltaNp73.

TP63, a member of the TP53 gene family, encodes two groups of three isoforms (alpha, beta and gamma). The TAp63 isoforms act as transcription factors. The DeltaNp63 isoforms lack the main transcription activation domain and act as dominant-negative inhibitors of transactivation (TA) isoforms. To clarify the role of these isoforms and to better understand their functional overlap with p53, we ectopically expressed each p63 isoform in the p53-null hepatocellular carcinoma cell line Hep3B. All TA isoforms, as well as DeltaNp63alpha, had a half-life of <1 h when transiently expressed and were degraded by the proteasome pathway. The most stable form was DeltaNp63gamma, with a half-life of >8 h. As expected, TA isoforms differed in their transcriptional activities toward genes regulated by p53, TAp63gamma being the most active form. In contrast, DeltaNp63 isoforms were transcriptionally inactive on genes studied and inhibited TA isoforms in a dose-dependent manner. When stably expressed in polyclonal cell populations, TAp63beta and gamma isoforms were undetectable. However, when treated with doxorubicin (DOX), p63 proteins rapidly accumulated in the cells. This stabilization was associated with an increase in phosphorylation. Strikingly, in DOX-treated polyclonal populations, increase in TAp63 levels was accompanied by overexpression of DeltaNp73. This observation suggests complex regulatory cross talks between the different isoforms of the p53 family. In conclusion, p63 exhibits several transcriptional and stress-response properties similar to those of p53, suggesting that p63 activities should be taken into consideration in approaches to improve cancer therapies based on genotoxic agents.

[Inactivation of failsafe programs by Twist oncoproteins and tumor progression]. / Inactivation des systèmes de sauvegarde cellulaire par les oncoprotéines Twist et progression tumorale.

Multicellular organisms have developed innate defense mechanisms to prevent the expansion of abnormal cells with significant proliferative potential. The two major safeguard mechanisms are premature senescence, which is characterized by definitive cell cycle arrest, and apoptosis, the most common form of programmed cell death. In normal and premalignant cells, the control of these processes is coupled to the regulation of cell proliferation, mainly through the p16 (Ink4A) -Rb and ARF-p53 intracellular signaling pathways. Hence, in benign tumors, aberrant mitogenic activity is counterbalanced by the induction of these oncosuppressive pathways, leading to either apoptosis or senescence which both limit tumor outgrowth. Progression towards malignant and potentially metastatic tumors requires the inhibition of these failsafe programs. Based on our work on Twist oncoproteins, we propose a presentation of recent data on cellular mechanisms by which cancer cells override the surveillance machinery and escape senescence and apoptosis, and we will describe the biological impact of this process on tumor metastasis.

TRF2 inhibition promotes anchorage-independent growth of telomerase-positive human fibroblasts.

Although telomere instability is observed in human tumors and is associated with the development of cancers in mice, it has yet to be established that it can contribute to the malignant transformation of human cells. We show here that in checkpoint-compromised telomerase-positive human fibroblasts an episode of TRF2 inhibition promotes heritable changes that increase the ability to grow in soft agar, but not tumor growth in nude mice. This transforming activity is associated to a burst of telomere instability but is independent of an altered control of telomere length. Moreover, it cannot be recapitulated by an increase in chromosome breaks induced by an exposure to gamma-radiations. Since it can be revealed in the context of telomerase-proficient human cells, telomere dysfunction might contribute to cancer progression even at late stages of the oncogenesis process, after the telomerase reactivation step.

Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens.

In lung and liver cancers, p53 mutations are mostly G:C to T:A transversions. This type of mutation is known to be induced by benzo(a)pyrene and aflatoxin B1 which are associated with the etiology of lung and liver cancers, respectively. Using a novel assay based on DNA polymerase fingerprint analysis, we identified p53 nucleotides targeted by these carcinogens. Thirteen of 14 nucleotide residues of the p53 gene which underwent G:C to T:A mutations in lung cancers were targeted by benzo(a)pyrene. Similarly, aflatoxin B1 formed adducts at a mutational hotspot specific for liver cancer. The same nucleotide (third base of codon 249), which mutates rarely in lung cancers, was not a target for benzo(a)pyrene. These in vitro observations indicate that p53 mutational hotspots identified in different tumors are selected targets specifically for the etiologically defined environmental carcinogens.

p53 induction prevents accumulation of aberrant transcripts in cancer cells.

Loss of fidelity of the splicing process occurs during tumor progression and can have a deleterious effect on genes like tumor suppressor genes. It was reported recently that the presence of aberrant transcripts of the TSG101 gene in breast cancer cells was associated with the mutation of the p53 tumor suppressor gene. On the basis of this observation, we have analyzed TSG101 transcript patterns in p53-active and p53-inactive cells. Using several isogenic cellular models, we demonstrate that the induction of p53 in cancer cells leads to a significant decrease of aberrant transcripts levels. This indicates a novel implication of p53 in the regulation of the splicing process.

Neurofibromatosis and early onset of cancers in hMLH1-deficient children.

Hereditary nonpolyposis colon cancer is a common hereditary disorder caused by the germ-line mutations of DNA mismatch repair (MMR) genes, especially hMLH1 and hMSH2. We report here the first identification of human compounds with a homozygous inactivation of a MMR gene. In a typical hereditary nonpolyposis colon cancer family, MMR-deficient children conceived from matings between heterozygotes for a hMLH1 deleterious mutation exhibited clinical features of de novo neurofibromatosis type I and early onset of extracolonic cancers. This observation demonstrates that MMR deficiency is compatible with human development but may lead to mutations during embryogenesis. On the basis of clinical symptoms observed in MMR-deficient children, we speculate that the neurofibromatosis type 1 gene is a preferential target for such alterations.

Hepatocarcinoma-specific mutant p53-249ser induces mitotic activity but has no effect on transforming growth factor beta 1-mediated apoptosis.

Mutations affecting the p53 gene abrogate its tumor suppressor activity. It is, however, unclear whether such mutations can generate mutant p53 proteins with an intrinsic transforming ability. More importantly, the mechanism(s) by which they exert such activity is unknown. We report here that p53-deficient hepatoma cells (Hep3B) transfected with mutant p53-249ser (codon 249 Arg-->Ser) acquire a new phenotype with an increased in vitro survival and mitotic activity. However, such a phenotypic change is not sufficient to cause a major shift in the poor tumorigenic potential of these cells. This is apparently due to transforming growth factor beta 1-mediated apoptotic death of Hep3B cells which is not affected by the expression of p53-249ser.

Prognostic value of P53 gene mutations in a large series of node-negative breast cancer patients.

The most important subgroup of breast cancer patients for which reliable prognostic factors are needed are women without axillary lymph node involvement. Although overall, these patients have a good prognosis, it is known that 20-30% will experience a recurrence of the disease. To determine the prognostic significance of P53 tumor suppressor gene mutation, specimens from 113 primary breast cancers were evaluated for the presence of P53 alterations, as detected by cDNA sequencing of the entire coding sequence of the gene. The median follow-up for patients was 105 months. P53 gene mutation was an independent prognostic marker of early relapse and death. Our results suggest that P53 gene mutations could be an important factor to identify node-negative patients who have a poor prognosis in the absence of adjuvant therapy. Prospective studies should be designed to determine which therapy should be performed in this subgroup of patients.

Detection of exon deletions and duplications of the mismatch repair genes in hereditary nonpolyposis colorectal cancer families using multiplex polymerase chain reaction of short fluorescent fragments.

Large genomic deletions within the mismatch repair MLH1 and MSH2 genes have been identified in families with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, and their detection represents a technical problem. To facilitate their detection, we developed a simple semiquantitative procedure based on the multiplex PCR of short fluorescent fragments. This method allowed us to confirm in HNPCC families three known deletions of MLH1 or MSH2 and to detect in 19 HNPCC families, in which analysis of mismatch repair genes using classical methods had revealed no alteration, a deletion of exon 5 and a duplication of MSH2 involving exons 9 and 10. The presence of such duplications, the frequency of which is probably underestimated, must be considered in HNPCC families in which conventional screening methods have failed to detect mutations.

[The enigma of the biological interpretation of the linear-quadratic model finally resolved? A summary for non-mathematicians]. / L'énigme de l'interprétation biologique du modèle linéaire-quadratique enfin résolue ? Une synthèse pour les non-mathématiciens.

The linear-quadratic (LQ) model is the only mathematical formula linking cellular survival and radiation dose that is sufficiently consensual to help radiation oncologists and radiobiologists in describing the radiation-induced events. However, this formula proposed in the 1970s and α and ß parameters on which it is based remained without relevant biological meaning. From a collection of cutaneous fibroblasts with different radiosensitivity, built over 12 years by more than 50 French radiation oncologists, we recently pointed out that the ATM protein, major actor of the radiation response, diffuses from the cytoplasm to the nucleus after irradiation. The evidence of this nuclear shuttling of ATM allowed us to provide a biological interpretation of the LQ model in its mathematical features, validated by a hundred of radiosensitive cases. A mechanistic explanation of the radiosensitivity of syndromes caused by the mutation of cytoplasmic proteins and of the hypersensitivity to low-dose phenomenon has been proposed, as well. In this review, we present our resolution of the LQ model in the most didactic way.

[Repeated radiation dose effect and DNA repair: Importance of the individual factor and the time interval between the doses]. / Effets de répétitions de doses d'irradiation et réparation de l'ADN : importance du facteur individuel et de l'intervalle de temps entre les doses.

The dose fractionation effect is a recurrent question of radiation biology research that remains unsolved since no model predicts the clinical effect only with the cumulated dose and the radiobiology of irradiated tissues. Such an important question is differentially answered in radioprotection, radiotherapy, radiology or epidemiology. A better understanding of the molecular response to radiation makes possible today a novel approach to identify the parameters that condition the fractionation effect. Particularly, the time between doses appears to be a key factor since it will permit, or not, the repair of certain radiation-induced DNA damages whose repair rates are of the order of seconds, minutes or hours: the fractionation effect will therefore vary according to the functionality of the different repair pathways, whatever for tumor or normal tissues.

p53 as a growth suppressor gene in HBV-related hepatocellular carcinoma cells.

Mutations of the p53 detected in human hepatocellular carcinomas suggest that its inactivation is a critical step in hepatocellular carcinogenesis. In order to test whether the expression of p53 is compatible with the transformed phenotype of hepatoma cells, we transfected Hep 3B cell line with p53 expression vectors. This cell line, which contains integrated hepatitis B virus sequences, is a good model to study whether the wild-type p53 can function as a tumour suppressor gene in virus-related hepatocellular carcinoma. Wild-type and mutant p53 (Ala143)-expression vectors containing a neoR gene were used. The number of antibiotic-resistant colonies was six times lower after transfection with wild-type p53 vector as compared to the mutant vector. As measured by a specific radioimmunoassay, six of eight (75%) colonies randomly selected after mutant p53 transfections expressed the transfected mutant p53 protein. In contrast, out of eight colonies from wild-type p53-transfections, none expressed detectable p53 protein. The absence of p53 protein was due to the selective deletion of transfected wild-type p53 cDNA sequences. These studies demonstrate that the growth of hepatocellular carcinoma cells is not compatible with the expression of wild-type p53. Therefore, p53 should be considered as a tumour suppressor gene in hepatocytes.

Absence of p53-dependent induction of the metastatic suppressor KAI1 gene after DNA damage.

The p53 tumor suppressor protein functions to monitor the integrity of the genome. If a damage is detected, p53 binds tightly to specific sequence elements in the DNA and induces the transactivation of genes involved in various growth regulatory processes such as cell cycle progression, DNA repair and apoptosis. A p53-binding site was recently identified in the promoter region of the metastatic suppressor KAI1 gene, suggesting that this gene was a direct transcriptional target of p53. To test the relevance of this hypothesis, we studied the endogenous KAI1 expression in a series of human cell lines with varying p53 status in response to genotoxic treatment as well as in different cellular models exhibiting an inducible p53 activity. Overall, our data indicate that KAI1 expression is not significantly modulated by p53. This observation provides a direct evidence that the presence of a p53-binding site in regulatory domains is not a sufficient criteria to define a p53-transcriptional target gene.

p21(WAF1/CIP1) response to genotoxic agents in wild-type TP53 expressing breast primary tumours.

Functional inactivation of the wild-type p53 protein has been described in different human cancers. Since a significant proportion of breast tumours express wild-type TP53, the p53 antiproliferative activity could be inactivated in transformed mammary epithelial cells by a mechanism independent on structural alteration of the gene. To test this hypothesis, we analysed the p53 activity in primary breast tumour cells. As a preliminary study, we demonstrated in breast adenocarcinoma cell lines that the nuclear accumulation of the inhibitor of cyclin dependent kinase p21(WAFl/CIP1), in response to adriamycin treatment, specifically reflected the activity of a functional wild-type p53 protein. Then, we used this strategy to study the p53 activity in 23 primary breast tumours. p21(WAF1/CIP1 accumulation was detected in all tumours expressing wild-type TP53. In contrast, no p21(WAF1/CIP1) response was detected in cells harboring a mutant TP53 gene. This report is the first functional study of p53 in primary breast tumours. The results demonstrate that TP53 mutation represents the only common mechanism leading to an irreversible inactivation of p53 functions in this cancer type.

Low frequency of TSG101/CC2 gene alterations in invasive human breast cancers.

Large intragenic deletions of the TSG101/CC2 gene were recently reported in seven of 15 primary metastatic breast cancers. Although the number of samples was small, this observation suggested that TSG101/CC2 alterations were a major event in breast carcinogenesis. To study the frequency of these deletions in invasive breast cancers we analysed 189 primary invasive breast tumours and 59 breast cancer metastases. We detected intragenic rearrangements in only three samples (two primary tumours and one metastasis). Northern blot analysis of 43 tumours without rearrangements failed to detect any abnormalities. Furthermore, we studied TSG101/CC2 in 11 human breast adenocarcinoma cell lines by Southern blot, RT-PCR and sequencing of the entire coding region of the gene, and detected no abnormalities. These results show that genetic alteration of TSG101/CC2 is a rare event in breast cancer.