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.

[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.

The emerging role of Twist proteins in hematopoietic cells and hematological malignancies.

Twist1 and Twist2 (Twist1-2) are two transcription factors, members of the basic helix-loop-helix family, that have been well established as master transcriptional regulators of embryogenesis and developmental programs of mesenchymal cell lineages. Their role in oncogenesis in epithelium-derived cancer and in epithelial-to-mesenchymal transition has also been thoroughly characterized. Recently, emerging evidence also suggests a key role for Twist1-2 in the function and development of hematopoietic cells, as well as in survival and development of numerous hematological malignancies. In this review, we summarize the latest data that depict the role of Twist1-2 in monocytes, T cells and B lymphocyte activation, and in associated hematological malignancies.

TP63 P2 promoter functional analysis identifies ß-catenin as a key regulator of ΔNp63 expression.

The ΔNp63 protein, a product of the TP63 gene that lacks the N-terminal domain, has a critical role in the maintenance of self renewal and progenitor capacity in several types of epithelial tissues. ΔNp63 is frequently overexpressed in squamous cell carcinoma (SCC) and in some other epithelial tumours. This overexpression may contribute to tumour progression through dominant-negative effects on the transcriptionally active (TA) isoforms of the p53 family (TAp63, TAp73 and p53), as well as through independent mechanisms. However, the molecular basis of ΔNp63 overexpression is not fully understood. Here, we show that the expression of ΔNp63 is regulated by the Wnt/ß-catenin pathway in human hepatocellular carcinoma (HCC) and SCC cell lines. This regulation operates in particular through TCF/LEF sites present in the P2 promoter of TP63. In addition, we show that ΔNp63 and ß-catenin are frequently coexpressed and accumulated in oesophageal SCC, but not in HCC. These results suggest that activation of the ß-catenin pathway may contribute to overexpression of ΔNp63 during tumour progression, in a cell type-specific manner.

Mutant p53(R175H) upregulates Twist1 expression and promotes epithelial-mesenchymal transition in immortalized prostate cells.

A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominant-negative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 'gain of function' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53(R175H) mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53(R175H), was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial-mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53(R175H) mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53(R175H) mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.

TWISTing an embryonic transcription factor into an oncoprotein.

Over the past decade, the reactivation of TWIST embryonic transcription factors has been described as a frequent event and a marker of poor prognosis in an impressive array of human cancers. Growing evidence now supports the premise that these cancers hijack TWIST's embryonic functions, granting oncogenic and metastatic properties. In this review, we report on the history and recent breakthroughs in understanding TWIST protein functions and the emerging role of the associated epithelial-mesenchymal transition (EMT) in tumorigenesis. We then broaden the discussion to address the general contribution of reactivating embryonic programs in cancerogenesis.

[Role of the epithelial-mesenchymal transition during tumor progression]. / Rôle de la transition épithéliomésenchymateuse dans la progression tumorale.

The epithelial-mesenchymal transition (EMT) is a morphogenetic program that converts epithelial into mesenchymal cells during the embryonic development. This mechanism is frequently reactivated during tumor progression and provides cells with motility and invasive capabilities favoring the metastatic dissemination from epithelial tumors. Various EMT-inducing transcription factors, such as the TWIST proteins, were also shown to inhibit oncogene-induced fail-safe programs (senescence and apoptosis), thereby promoting the progression from benign to malignant stages. Altogether, these observations suggest that EMT could play an important role in favoring both tumor development and metastatic dissemination.

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.

[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.

Lobular and ductal carcinomas of the breast have distinct genomic and expression profiles.

Invasive ductal carcinomas (IDCs) and invasive lobular carcinomas (ILCs) are the two major pathological types of breast cancer. Epidemiological and histoclinical data suggest biological differences, but little is known about the molecular alterations involved in ILCs. We undertook a comparative large-scale study by both array-compared genomic hybridization and cDNA microarray of a set of 50 breast tumors (21 classic ILCs and 29 IDCs) selected on homogeneous histoclinical criteria. Results were validated on independent tumor sets, as well as by quantitative RT-PCR. ILCs and IDCs presented differences at both the genomic and expression levels with ILCs being less rearranged and heterogeneous than IDCs. Supervised analysis defined a 75-BACs signature discriminating accurately ILCs from IDCs. Expression profiles identified two subgroups of ILCs: typical ILCs ( approximately 50%), which were homogeneous and displayed a normal-like molecular pattern, and atypical ILCs, more heterogeneous with features intermediate between ILCs and IDCs. Supervised analysis identified a 75-gene expression signature that discriminated ILCs from IDCs, with many genes involved in cell adhesion, motility, apoptosis, protein folding, extracellular matrix and protein phosphorylation. Although ILCs and IDCs share common alterations, our data show that ILCs and IDCs could be distinguished on the basis of their genomic and expression profiles suggesting that they evolve along distinct genetic pathways.

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.

Paclitaxel resistance in untransformed human mammary epithelial cells is associated with an aneuploidy-prone phenotype.

Despite its increasing clinical use, almost no data are currently available about paclitaxel effects on non-cancerous mammary epithelial cells. We have previously established paclitaxel-resistant sub-cell lines (paclitaxel-surviving populations, PSPs; n=20), and sensitive controls (control clones, CCs; n=10), from the untransformed human mammary epithelial cell line HME1. In this study, we aimed to establish whether paclitaxel resistance was associated with a modified sensitivity to paclitaxel-induced aneuploidy. For this purpose, we analysed basal and paclitaxel-induced chromosome missegregation, apoptosis and aberrant spindle multipolarisation as well as microtubular network composition for each subline. PSP sublines showed higher basal and paclitaxel-induced chromosome missegregation than the CC sublines. This phenomenon was associated with resistance to paclitaxel-induced apoptosis. No significant difference in paclitaxel-induced spindle pole abnormalities between CC and PSP sublines was found. Besides, we showed that a majority of PSPs display a constitutively disrupted microtubular network composition due to aberrant tubulin expression and post-translational modifications. These results clearly indicate that paclitaxel resistance in untransformed human mammary epithelial cells is related to an increased susceptibility to acquire aneuploidy in response to this agent. The consequences of these paclitaxel-associated alterations could be deleterious as they can potentially trigger tumorigenesis.

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.

A twist for survival and cancer progression.

A major obstacle to the expansion of abnormal cells with significant proliferative potential is the induction of programmed cell death. Consequently, oncogene-driven hyperproliferation must be associated with apoptosis inhibition to allow malignant outgrowth. The oncogenic cooperation of N-Myc and Twist-1 in the development of neuroblastoma, the most common and deadly solid tumour of childhood, perfectly illustrates such a process. N-Myc promotes cell proliferation, whereas Twist-1 counteracts its pro-apoptotic properties by knocking-down the ARF/p53 pathway. On the basis of numerous recent studies reporting its overexpression in a variety of human cancers, we discuss in this review the role of Twist-1 as a potent inhibitor of the cell safety programs engaged in response to an abnormal mitogenic activity.

Drug resistance associated with loss of p53 involves extensive alterations in microtubule composition and dynamics.

In the present study, we compared the dynamics and composition of microtubules in cell lines derived from the human breast adenocarcinoma MCF-7 containing either the wild-type p53 (wt-p53; MN1) or a dominant-negative variant of p53 gene (mut-p53; MDD2). Mut-p53 cells were significantly resistant to the cytotoxicity of the microtubule-targeted drugs (vinca alkaloids and taxanes), as compared with wt-p53 cells. Studies by high-resolution time-lapse fluorescence microscopy in living cells indicated that the dynamics of microtubules of mut-p53 cells were altered in complex ways and were significantly increased as compared with microtubules in wt-p53 cells. The percentage of time microtubules spent in growing and shortening phases increased significantly, their catastrophe frequency increased, and their overall dynamicity increased by 33%. In contrast, their shortening rate and the mean length shortened decreased. Cells containing mut-p53 displayed increased polymerisation of tubulin, increased protein levels of the class IV beta-tubulin isotype, STOP and survivin, and reduced protein levels of class II beta-tubulin isotype, MAP4 and FHIT. We conclude that p53 protein may contribute to the regulation of microtubule composition and function, and that alterations in p53 function may generate complex microtubule-associated mechanisms of resistance to tubulin-binding agents.

Inactivation of wild-type p53 by a dominant negative mutant renders MCF-7 cells resistant to tubulin-binding agent cytotoxicity.

The present study was performed to gain insight into the role of p53 on the cytotoxicity of tubulin-binding agents (TBA) on cancer cells. Drug sensitivity, cell cycle distribution and drug-induced apoptosis were compared in 2 lines derived from the mammary adenocarcinoma MCF-7: the MN-1 cell line containing wild-type p53 (wt-p53) and the MDD2 line, containing a dominant negative variant of the p53 protein (mut-p53). The MDD2 cell line was significantly more resistant to the cytotoxic effects of vinblastine and paclitaxel than the MN1 cell line. MN1 cells, but not MDD2 cells, displayed wt-p53 protein accumulation as well as p21/WAF1 and cyclin G1 induction after exposure to TBA. Both cell lines arrested at G(2)/M after drug treatment. However exposure of MN1 cells to TBA resulted in a stronger variation in mitochondrial membrane potential, associated with cleavage of PARP, and more apoptosis, as measured by annexin V expression. After exposure to vinblastine, Raf 1 kinase activity was reduced in MDD2 cells but not in MN1 cells. Addition of flavopiridol to vinblastine- and paclitaxel-treated cells reversed the MDD2-resistant phenotype by inducing G(1)cell cycle arrest and inhibiting endoreduplication. We conclude that the p53 status of cancer cells influences their sensitivity to TBA cytotoxicity. This effect is likely to involve differences in the apoptotic cascade.

Absence of rearrangements in the BRCA2 gene in human cancers.

Mutations of BRCA2 in sporadic breast and ovarian carcinomas are exceedingly rare. This led to the suggestion that large genomic rearrangements could be involved. We performed Southern blots in genomic DNA from 130 primary breast cancers and 83 cancer cell lines (breast, ovarian, pancreatic and small cell lung carcinomas) and found no genomic rearrangements. These results suggest that a gene other than BRCA2 is the target of the frequent 13q12.3 allelic deletions in human cancers.