Radiotherapy orchestrates natural killer cell dependent antitumor immune responses through CXCL8.

Radiotherapy is a mainstay cancer therapy whose antitumor effects partially depend on T cell responses. However, the role of Natural Killer (NK) cells in radiotherapy remains unclear. Here, using a reverse translational approach, we show a central role of NK cells in the radiation-induced immune response involving a CXCL8/IL-8-dependent mechanism. In a randomized controlled pancreatic cancer trial, CXCL8 increased under radiotherapy, and NK cell positively correlated with prolonged overall survival. Accordingly, NK cells preferentially infiltrated irradiated pancreatic tumors and exhibited CD56dim-like cytotoxic transcriptomic states. In experimental models, NF-κB and mTOR orchestrated radiation-induced CXCL8 secretion from tumor cells with senescence features causing directional migration of CD56dim NK cells, thus linking senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined high-dose radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice, suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy.

Combined inhibition of CXCL12 and PD-1 in MSS colorectal and pancreatic cancer: modulation of the microenvironment and clinical effects.

BACKGROUND: Immunotherapy in microsatellite stable colorectal or pancreatic cancer has not shown promising results. It has been hypothesized that targeting immunosuppressive molecules like SDF1-alpha/CXCL12 could contribute to immunotherapy and animal models showed promising results on T cell activation and migration in combination with immune checkpoint inhibition. METHODS: Here, we describe the successful application of anti-CXCL12 (NOX-A12) in patients with advanced stage pretreated metastatic colorectal and pancreatic cancer (OPERA trial). The treatment consisted of 2 weeks of anti-CXCL12 monotherapy with NOX-A12 followed by combination therapy with pembrolizumab (n=20 patients) until progression or intolerable toxicity had occurred. RESULTS: The treatment was safe and well tolerated with 83.8% grade I/II, 15.5% grade III and 0.7% grade V adverse events. Of note, for a majority of patients, time on trial treatment was prolonged compared with their last standard treatment preceding trial participation. Systematic serial biopsies revealed distinct patterns of modulation. Tissue and clinical responses were associated with Th1-like tissue reactivity upon CXCL12 inhibition. A downregulation of a cytokine cassette of interleukin (IL)-2/IL-16/CXCL-10 was associated with tumor resistance and furthermore linked to a rare, CXCL12-associated CD14+CD15+promonocytic population. T cells showed aggregation and directed movement towards the tumor cells in responding tissues. Serum analyses detected homogeneous immunomodulatory patterns in all patients, regardless of tissue responses. CONCLUSIONS: We demonstrate that the combination of CXCL12 inhibition and checkpoint inhibition is safe and grants further exploration of synergistic combinatorial strategies.

CCR5 status and metastatic progression in colorectal cancer.

Multiple reports have highlighted the importance of the local immunological cellular composition (i.e. the density of effector T cells and macrophage polarization state) in predicting clinical outcome in advanced metastatic stage of colorectal cancer. However, in spite of the general association between a high effector T cell density and improved outcome, our recent work has revealed a specific lymphocyte-driven cancer cell-supporting signal. Indeed, lymphocyte-derived CCL5 supports CCR5-positive tumor cell proliferation and thereby fosters tumor growth in metastatic liver lesions. Upon systematic analysis of CCR5 expression by tumor cells using immunohistochemistry, we observed that the intensity of CCR5 increases with primary tumor size and peaks in T4 tumors. In liver metastases however, though CCR5 expression intensity is globally heightened compared to primary tumors, alterations in the expression patterns appear, leading to "patchiness" of the stain. CCR5 patchiness is, therefore, a signature of liver metastases in our cohort (n = 97 specimens) and relates to globally decreased expression intensity, but does not influence the extent of the response to CCR5 inhibitor Maraviroc in patients. Moreover, CCR5 patchiness relates to a poor immune landscape characterized by a low cytotoxic-to-regulatory T cell ratio at the invasive margin and enriched cellular and molecular markers of macrophage M2 polarization. Finally, because higher numbers of PD-1- and CTLA-4-positive cells surround tumors with patchy CCR5 expression, one can speculate that these tumors potentially respond to immune checkpoint blockade. This hypothesis is corroborated by the prolonged disease-free survival and disease-specific survival observed in patients with low gene expression of CCR5 in metastases from two publically available cohorts. These observations highlight the complex role of the CCL5-CCR5 axis in CRC metastatic progression and warrant further investigations.

Topography of cancer-associated immune cells in human solid tumors.

Lymphoid and myeloid cells are abundant in the tumor microenvironment, can be quantified by immunohistochemistry and shape the disease course of human solid tumors. Yet, there is no comprehensive understanding of spatial immune infiltration patterns ('topography') across cancer entities and across various immune cell types. In this study, we systematically measure the topography of multiple immune cell types in 965 histological tissue slides from N = 177 patients in a pan-cancer cohort. We provide a definition of inflamed ('hot'), non-inflamed ('cold') and immune excluded patterns and investigate how these patterns differ between immune cell types and between cancer types. In an independent cohort of N = 287 colorectal cancer patients, we show that hot, cold and excluded topographies for effector lymphocytes (CD8) and tumor-associated macrophages (CD163) alone are not prognostic, but that a bivariate classification system can stratify patients. Our study adds evidence to consider immune topographies as biomarkers for patients with solid tumors.

High-Throughput Screening of Combinatorial Immunotherapies with Patient-Specific In Silico Models of Metastatic Colorectal Cancer.

Solid tumors are rich ecosystems of numerous different cell types whose interactions lead to immune escape and resistance to immunotherapy in virtually all patients with metastatic cancer. Here, we have developed a 3D model of human solid tumor tissue that includes tumor cells, fibroblasts, and myeloid and lymphoid immune cells and can represent over a million cells over clinically relevant timeframes. This model accurately reproduced key features of the tissue architecture of human colorectal cancer and could be informed by individual patient data, yielding in silico tumor explants. Stratification of growth kinetics of these explants corresponded to significantly different overall survival in a cohort of patients with metastatic colorectal cancer. We used the model to simulate the effect of chemotherapy, immunotherapies, and cell migration inhibitors alone and in combination. We classified tumors according to tumor and host characteristics, showing that optimal treatment strategies markedly differed between these classes. This platform can complement other patient-specific ex vivo models and can be used for high-throughput screening of combinatorial immunotherapies.Significance: This patient-informed in silico tumor growth model allows testing of different cancer treatment strategies and immunotherapies on a cell/tissue level in a clinically relevant scenario. Cancer Res; 78(17); 5155-63. ©2018 AACR.

Large-scale database mining reveals hidden trends and future directions for cancer immunotherapy.

Cancer immunotherapy has fundamentally changed the landscape of oncology in recent years and significant resources are invested into immunotherapy research. It is in the interests of researchers and clinicians to identify promising and less promising trends in this field in order to rationally allocate resources. This requires a quantitative large-scale analysis of cancer immunotherapy related databases. We developed a novel tool for text mining, statistical analysis and data visualization of scientific literature data. We used this tool to analyze 72002 cancer immunotherapy publications and 1469 clinical trials from public databases. All source codes are available under an open access license. The contribution of specific topics within the cancer immunotherapy field has markedly shifted over the years. We show that the focus is moving from cell-based therapy and vaccination towards checkpoint inhibitors, with these trends reaching statistical significance. Rapidly growing subfields include the combination of chemotherapy with checkpoint blockade. Translational studies have shifted from hematological and skin neoplasms to gastrointestinal and lung cancer and from tumor antigens and angiogenesis to tumor stroma and apoptosis. This work highlights the importance of unbiased large-scale database mining to assess trends in cancer research and cancer immunotherapy in particular. Researchers, clinicians and funding agencies should be aware of quantitative trends in the immunotherapy field, allocate resources to the most promising areas and find new approaches for currently immature topics.

In Silico Modeling of Immunotherapy and Stroma-Targeting Therapies in Human Colorectal Cancer.

Despite the fact that the local immunological microenvironment shapes the prognosis of colorectal cancer, immunotherapy has shown no benefit for the vast majority of colorectal cancer patients. A better understanding of the complex immunological interplay within the microenvironment is required. In this study, we utilized wet lab migration experiments and quantitative histological data of human colorectal cancer tissue samples (n = 20) including tumor cells, lymphocytes, stroma, and necrosis to generate a multiagent spatial model. The resulting data accurately reflected a wide range of situations of successful and failed immune surveillance. Validation of simulated tissue outcomes on an independent set of human colorectal cancer specimens (n = 37) revealed the model recapitulated the spatial layout typically found in human tumors. Stroma slowed down tumor growth in a lymphocyte-deprived environment but promoted immune escape in a lymphocyte-enriched environment. A subgroup of tumors with less stroma and high numbers of immune cells showed high rates of tumor control. These findings were validated using data from colorectal cancer patients (n = 261). Low-density stroma and high lymphocyte levels showed increased overall survival (hazard ratio 0.322, P = 0.0219) as compared with high stroma and high lymphocyte levels. To guide immunotherapy in colorectal cancer, simulation of immunotherapy in preestablished tumors showed that a complex landscape with optimal stroma permeabilization and immune cell activation is able to markedly increase therapy response in silico These results can help guide the rational design of complex therapeutic interventions, which target the colorectal cancer microenvironment. Cancer Res; 77(22); 6442-52. ©2017 AACR.

EMT and inflammation: inseparable actors of cancer progression.

Tumors can be depicted as wounds that never heal, and are infiltrated by a large array of inflammatory and immune cells. Tumor-associated chronic inflammation is a hallmark of cancer that fosters progression to a metastatic stage, as has been extensively reviewed lately. Indeed, inflammatory cells persisting in the tumor establish a cross-talk with tumor cells that may result in a phenotype switch into tumor-supporting cells. This has been particularly well described for macrophages and is referred to as tumor-associated 'M2' polarization. Epithelial-to-mesenchymal transition (EMT), the embryonic program that loosens cell-cell adherence complexes and endows cells with enhanced migratory and invasive properties, can be co-opted by cancer cells during metastatic progression. Cancer cells that have undergone EMT are more aggressive, displaying increased invasiveness, stem-like features, and resistance to apoptosis. EMT programs can also stimulate the production of proinflammatory factors by cancer cells. Conversely, inflammation is a potent inducer of EMT in tumors. Therefore, the two phenomena may sustain each other, in an alliance for metastasis. This is the focus of this review, where the interconnections between EMT programs and cellular and molecular actors of inflammation are described. We also recapitulate data linking the EMT/inflammation axis to metastasis.

TGFBR2-dependent alterations of exosomal cargo and functions in DNA mismatch repair-deficient HCT116 colorectal cancer cells.

BACKGROUND: Colorectal cancers (CRCs) that lack DNA mismatch repair function exhibit the microsatellite unstable (MSI) phenotype and are characterized by the accumulation of frameshift mutations at short repetitive DNA sequences (microsatellites). These tumors recurrently show inactivating frameshift mutations in the tumor suppressor Transforming Growth Factor Beta Receptor Type 2 (TGFBR2) thereby abrogating downstream signaling. How altered TGFBR2 signaling affects exosome-mediated communication between MSI tumor cells and their environment has not been resolved. Here, we report on molecular alterations of exosomes shed by MSI cells and the biological response evoked in recipient cells. METHODS: Exosomes were isolated and characterized by electron microscopy, nanoparticle tracking, and western blot analysis. TGFBR2-dependent effects on the cargo and functions of exosomes were studied in a MSI CRC model cell line enabling reconstituted and inducible TGFBR2 expression and signaling. Microsatellite frameshift mutations in exosomal and cellular DNA were examined by PCR-based DNA fragment analysis and exosomal protein profiles were identified by mass spectrometry. Uptake of fluorescent-labeled exosomes by hepatoma recipient cells was monitored by confocal microscopy. TGFBR2-dependent exosomal effects on secreted cytokine levels of recipient cells were analyzed by Luminex technology and ELISA. RESULTS: Frameshift mutation patterns in microsatellite stretches of TGFBR2 and other MSI target genes were found to be reflected in the cargo of MSI CRC-derived exosomes. At the proteome level, reconstituted TGFBR2 expression and signaling uncovered two protein subsets exclusively occurring in exosomes derived from TGFBR2-deficient (14 proteins) or TGFBR2-proficient (five proteins) MSI donor cells. Uptake of these exosomes by recipient cells caused increased secretion (2-6 fold) of specific cytokines (Interleukin-4, Stem Cell Factor, Platelet-derived Growth Factor-B), depending on the TGFBR2 expression status of the tumor cell. CONCLUSION: Our results indicate that the coding MSI phenotype of DNA mismatch repair-deficient CRC cells is maintained in their exosomal DNA. Moreover, we uncovered that a recurrent MSI tumor driver mutation like TGFBR2 can reprogram the protein content of MSI cell-derived exosomes and in turn modulate the cytokine secretion profile of recipient cells. Apart from its diagnostic potential, these TGFBR2-dependent exosomal molecular and proteomic signatures might help to understand the signaling routes used by MSI tumors. Fricke et al. uncovered coding microsatellite instability-associated mutations of colorectal tumor driver genes like TGFBR2 in MSI tumor cellderived exosomes. Depending on the TGFBR2 expression status of their donor cells, shed exosomes show distinct proteomic signatures and promote altered cytokine secretion profiles in recipient cells.

Zonula occludens-1/NF-κB/CXCL8: a new regulatory axis for tumor angiogenesis.

Zonula occludens-1 (ZO-1) is a submembrane scaffolding protein that may display proinvasive functions when it relocates from tight junctions into the cytonuclear compartment. This article examines the functional involvement of ZO-1 in CXCL8/IL-8 chemokine expression in lung and breast tumor cells. ZO-1 small interfering RNA and cDNA transfection experiments emphasized regulation of CXCL8/IL-8 expression via a cytonuclear pool of ZO-1. Luciferase reporter assays highlighted a 173-bp region of CXCL8/IL-8 promoter that responded to ZO-1. Moreover, by using mutated promoter constructs, we identified a NF-κB site as critical in this activation. Furthermore, NF-κB pathway signaling analysis revealed both IκBα and p65 phosphorylation in ZO-1-overexpressing cells, and subsequent p65 silencing validated its requirement for CXCL8/IL-8 induction. Investigation of the functional implication of this regulatory axis next showed the proangiogenic activity of ZO-1 in both ex vivo and in vivo angiogenesis assays. Finally, we found that non-small-cell lung carcinoma that presented a cytonuclear ZO-1 pattern was significantly more angiogenic that that without detectable cytonuclear ZO-1 expression. Taken together, our results demonstrate that ZO-1 regulates CXCL8/IL-8 expression via the NF-κB signaling pathway and its p65 subunit, which subsequently modulates the transcription of IL-8. We also provide evidence of a newly identified regulatory pathway that could promote angiogenesis. Thus, our results support the concept that the ZO-1 shuttle from the cell junction to the cytonuclear compartment may affect both the intrinsic invasive properties of tumor cells and the establishment of the protumoral microenvironment.-Lesage, J., Suarez-Carmona, M., Neyrinck-Leglantier, D., Grelet, S., Blacher, S., Hunziker, W., Birembaut, P., Noël, A., Nawrocki-Raby, B., Gilles, C., Polette, M. Zonula occludens-1/NF-κB/CXCL8: a new regulatory axis for tumor angiogenesis.

Proteomic signatures reveal a dualistic and clinically relevant classification of anal canal carcinoma.

Aetiologically linked to HPV infection, malignancies of the anal canal have substantially increased in incidence over the last 20 years. Although most anal squamous cell carcinomas (SCCs) respond well to chemoradiotherapy, about 30% of patients experience a poor outcome, for undetermined reasons. Despite cumulative efforts for discovering independent predictors of overall survival, both nodal status and tumour size are still the only reliable factors predicting patient outcome. Recent efforts have revealed that the biology of HPV-related lesions in the cervix is strongly linked to the originally infected cell population. To address the hypothesis that topography also influences both gene expression profile and behaviour of anal (pre)neoplastic lesions, we correlated both proteomic signatures and clinicopathological features of tumours arising from two distinct portions of the anal canal: the lower part (squamous zone) and the more proximal anal transitional zone. Although microdissected cancer cells appeared indistinguishable by morphology (squamous phenotype), unsupervised clustering analysis of the whole proteome significantly highlighted the heterogeneity that exists within anal canal tumours. More importantly, two region-specific subtypes of SCC were revealed. The expression profile (sensitivity/specificity) of several selected biomarkers (keratin filaments) further confirmed the subclassification of anal (pre)cancers based on their cellular origin. Less commonly detected compared to their counterparts located in the squamous mucosa, SCCs originating in the transitional zone more frequently displayed a poor or basaloid differentiation, and were significantly correlated with reduced disease-free and overall survivals. Taken together, we present direct evidence that anal canal SCC comprises two distinct entities with different cells of origin, proteomic signatures, and survival rates. This study forms the basis for a dualistic classification of anal carcinoma, with implications for management, outcome expectations, and possibly therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tissue Factor Induced by Epithelial-Mesenchymal Transition Triggers a Procoagulant State That Drives Metastasis of Circulating Tumor Cells.

Epithelial-mesenchymal transition (EMT) is prominent in circulating tumor cells (CTC), but how it influences metastatic spread in this setting is obscure. Insofar as blood provides a specific microenvironment for tumor cells, we explored a potential link between EMT and coagulation that may provide EMT-positive CTCs with enhanced colonizing properties. Here we report that EMT induces tissue factor (TF), a major cell-associated initiator of coagulation and related procoagulant properties in the blood. TF blockade by antibody or shRNA diminished the procoagulant activity of EMT-positive cells, confirming a functional role for TF in these processes. Silencing the EMT transcription factor ZEB1 inhibited both EMT-associated TF expression and coagulant activity, further strengthening the link between EMT and coagulation. Accordingly, EMT-positive cells exhibited a higher persistance/survival in the lungs of mice colonized after intravenous injection, a feature diminished by TF or ZEB1 silencing. In tumor cells with limited metastatic capability, enforcing expression of the EMT transcription factor Snail increased TF, coagulant properties, and early metastasis. Clinically, we identified a subpopulation of CTC expressing vimentin and TF in the blood of metastatic breast cancer patients consistent with our observations. Overall, our findings define a novel EMT-TF regulatory axis that triggers local activation of coagulation pathways to support metastatic colonization of EMT-positive CTCs. Cancer Res; 76(14); 4270-82. ©2016 AACR.

Tumoral Immune Cell Exploitation in Colorectal Cancer Metastases Can Be Targeted Effectively by Anti-CCR5 Therapy in Cancer Patients.

The immune response influences the clinical course of colorectal cancer (CRC). Analyzing the invasive margin of human CRC liver metastases, we identified a mechanism of immune cell exploitation by tumor cells. While two distinct subsets of myeloid cells induce an influx of T cells into the invasive margin via CXCL9/CXCL10, CCL5 is produced by these T cells and stimulates pro-tumoral effects via CCR5. CCR5 blockade in patient-derived functional in vitro organotypic culture models showed a macrophage repolarization with anti-tumoral effects. These anti-tumoral effects were then confirmed in a phase I trial with a CCR5 antagonist in patients with liver metastases of advanced refractory CRC. Mitigation of tumor-promoting inflammation within the tumor tissue and objective tumor responses in CRC were observed.

Human Papillomavirus-related tumours of the oropharynx display a lower tumour hypoxia signature.

OBJECTIVES: Human Papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OSCC) patients have improved prognosis compared to other head and neck (HNSCC) cancers. Since poor prognosis is associated with tumour hypoxia, we studied whether the hypoxic response is different in HPV-related cells and tumours. MATERIAL AND METHODS: HPV-positive and -negative cells were incubated in hypoxia and analyzed by qRTPCR, western blotting and cell proliferation assays. Tumours formed by xenografting these cells in nude mice were studied by IHC. HNSCC patient samples were analyzed by unsupervised clustering of hypoxia-related gene expression, quantitative real-time PCR (qRTPCR) and immunohistochemical (IHC) detection of neo-blood vessels. RESULTS AND CONCLUSION: HPV-positive and -negative cells responded differently to hypoxia, in terms of gene expression (HIF-1α, PHD-3, GLUT-1 and VEGF-A) and cell survival. Tumour xenografts formed by HPV-positive cells had fewer hypoxic areas than those formed by HPV-negative cells. HPV related tumours were less hypoxic, expressed lower levels of hypoxia-responsive genes, and had a higher density of neo-blood vessels. HPV-related OSCC display lower tumour hypoxia, which could be linked to the distinct intrinsic abilities of HPV-positive tumour cells to adapt to hypoxia and to their better prognosis.

Soluble factors regulated by epithelial-mesenchymal transition mediate tumour angiogenesis and myeloid cell recruitment.

Epithelial-mesenchymal transition (EMT) programmes provide cancer cells with invasive and survival capacities that might favour metastatic dissemination. Whilst signalling cascades triggering EMT have been extensively studied, the impact of EMT on the crosstalk between tumour cells and the tumour microenvironment remains elusive. We aimed to identify EMT-regulated soluble factors that facilitate the recruitment of host cells in the tumour. Our findings indicate that EMT phenotypes relate to the induction of a panel of secreted mediators, namely IL-8, IL-6, sICAM-1, PAI-1 and GM-CSF, and implicate the EMT-transcription factor Snail as a regulator of this process. We further show that EMT-derived soluble factors are pro-angiogenic in vivo (in the mouse ear sponge assay), ex vivo (in the rat aortic ring assay) and in vitro (in a chemotaxis assay). Additionally, conditioned medium from EMT-positive cells stimulates the recruitment of myeloid cells. In a bank of 40 triple-negative breast cancers, tumours presenting features of EMT were significantly more angiogenic and infiltrated by a higher quantity of myeloid cells compared to tumours with little or no EMT. Taken together, our results show that EMT programmes trigger the expression of soluble mediators in cancer cells that stimulate angiogenesis and recruit myeloid cells in vivo, which might in turn favour cancer spread.

Carcinogenic HPV infection in the cervical squamo-columnar junction.

Recent studies have suggested the involvement of a unique population of cells at the cervical squamo-columnar junction (SCJ) in the pathogenesis of early (squamous intraepithelial lesion or SIL) and advanced (squamous cell and adeno-carcinomas) cervical neoplasia. However, there is little evidence to date showing that SCJ cells harbour carcinogenic HPV or are instrumental in the initial phases of neoplasia. This study was designed to (1) determine if normal-appearing SCJ cells contained evidence of carcinogenic HPV infection and (2) trace their transition to early SIL. Sections of cervix from high-risk reproductive age women were selected and SCJ cells were analysed by using several techniques which increasingly implicated HPV infection: HPV DNA (genotyping and in situ hybridization)/RNA (PCR), immunostaining for HPV16 E2 (an early marker of HPV infection), p16(ink4), Ki67, and HPV L1 protein. In 22 cases with a history of SIL and no evidence of preneoplastic lesion in the excision specimen, HPV DNA was isolated from eight of ten with visible SCJ cells, six of which were HPV16/18 DNA-positive. In five of these latter cases, the SCJ cells were positive for p16(ink4) and/or HPV E2. Transcriptionally active HPV infection (E6/E7 mRNAs) was also detected in microdissected SCJ cells. Early squamous atypia associated with the SCJ cells demonstrated in addition diffuse p16(ink4) immunoreactivity, elevated proliferative index, and rare L1 antigen positivity. We present for the first time direct evidence that normal-appearing SCJ cells can be infected by carcinogenic HPV. They initially express HPV E2 and their progression to SIL is heralded by an expanding metaplastic progeny with increased proliferation and p16(ink4) expression. Whether certain SCJs are more vulnerable than others to carcinogenic HPV genotypes and what variables determine transition to high-grade SIL remain unresolved, but the common event appears to be a vulnerable cell at the SCJ.

Defensins: "Simple" antimicrobial peptides or broad-spectrum molecules?

Small cationic peptides highly conserved in vertebrates, both α- and ß-defensins were primarily identified as anti-microbial compounds involved in innate immunity. While human α-defensins are mostly expressed by neutrophils, ß-defensins are secreted by epithelial cells of the skin and mucosae. Besides their anti-microbial activity, accumulating data emerged in the past decade indicating that defensins have extended functions in human physio(patho)logy. Indeed, defensins appeared as modulators of the adaptive immune system and angiogenesis, key mediators of wound healing and determinant players in male fertility. Furthermore, the impact of defensin expression in cancer and the potential use of these small peptides as biomarkers or even therapeutic tools should not be ignored. In the present review, we describe recent research works regarding the diversified functions of defensins, by mainly focusing on human models.

Unique recurrence patterns of cervical intraepithelial neoplasia after excision of the squamocolumnar junction.

Recent studies have identified a putative cell of origin for cervical intraepithelial neoplasia (CIN) and cervical cancer at the squamocolumnar junction (SCJ) and suggest that these cells may not regenerate after excision (loop electrosurgical excision procedure). Our study addressed the impact of SCJ excision on the temporal dynamics, histologic and viral (human papillomavirus, HPV) characteristics of recurrent CIN. One hundred and thirty-one consecutive patients treated by excision and attending follow-up visits were enrolled. We compared recurrent and initial CIN with attention to excision margins, timing of recurrence, CIN grade, HPV types, p16 immunophenotype and SCJ immunophenotype. During the follow-up period (up to 4 years), 16 (12.2%) recurrences were identified. Four (25%) were identified at the first follow-up visit, closely resembled the initial CIN 2/3 in grade and HPV type and were typically SCJ marker positive [SCJ(+)], suggesting nonexcised (residual) disease. Twelve (75%) manifested after the first postoperative visit and all were in the ectocervix or in mature metaplastic epithelium. All of the 12 delayed recurrences were classified as CIN 1 and were SCJ (-). In total, 9 out of 11 SCJ (-) recurrences (82%) followed regressed spontaneously. Taken together, these results show that new lesions developing from any HPV infection are delayed and occur within the ectocervix or metaplastic epithelium. This markedly lower risk of CIN 2/3 after successful SCJ excision suggests that the removal of the SCJ could be a critical variable in reducing the risk of subsequent CIN 2/3 and cervical cancer.

ΔNp63 isoform-mediated ß-defensin family up-regulation is associated with (lymph)angiogenesis and poor prognosis in patients with squamous cell carcinoma.

Beside a role in normal development/differentiation, high p63 immunoreactivity is also frequently observed in squamous cell carcinoma (SCC). Due to the complexity of the gene, the role of each p63 isotype in tumorigenesis is still confusing. Constitutively produced or induced in inflammatory conditions, human beta-defensins (HßDs) are cationic peptides involved in host defenses against bacteria, viruses and fungi. Here, we investigated both the role of p63 proteins in the regulation of HßDs and the implication of these antimicrobial peptides in tumor (lymph)angiogenesis. Thus, in contrast to TAp63 isotypes, we observed that ΔNp63 proteins (α, ß, γ) induce HßD1, 2 and 4 expression. Similar results were observed in cancer tissues and cell lines. We next demonstrated that ΔNp63-overexpressing SCC are associated with both a poor prognosis and a high tumor vascularisation and lymphangiogenesis. Moreover, we showed that HßDs exert a chemotactic activity for (lymphatic) endothelial cells in a CCR6-dependent manner. The ability of HßDs to enhance (lymph)angiogenesis in vivo was also evaluated. We observed that HßDs increase the vessel number and induce a significant increase in relative vascular area compared to negative control. Taken together, the results of this study suggest that ΔNp63-regulated HßD could promote tumor (lymph)angiogenesis in SCC microenvironment.

Regulation of p63 isoforms by snail and slug transcription factors in human squamous cell carcinoma.

TP63 is a p53-related gene that contains two alternative promoters, which give rise to transcripts that encode proteins with (TAp63) or without (DeltaNp63) an amino-transactivating domain. Whereas the expression of p63 is required for proper development of epithelial structures, the role of p63 in tumorigenesis remains unclear. Here, we investigated the role of Snail and Slug transcription factors, known to promote epithelial-to-mesenchymal transitions during development and cancer, in the regulation of p63 isoforms in human squamous cell carcinoma (SCC). In the present study, we observed that the expressions of DeltaN and TAp63 isoforms were, respectively, down- and up-regulated by both Snail and Slug. However, the induction of TAp63 was not directly caused by these two transcription factors but resulted from the loss of DeltaNp63, which acts as dominant-negative inhibitor of TAp63. In SCC cell lines and cancer tissues, high expression of Snail and Slug was also significantly associated with altered p63 expression. Finally, we showed that DeltaNp63 silencing reduced cell-cell adhesion and increased the migratory properties of cancer cells. These data suggest that the disruption of p63 expression induced by Snail and Slug plays a crucial role in tumor progression. Therefore, p63 and its regulating factors could constitute novel prognosis markers in patients with SCC and attractive targets for the therapeutic modulation of neoplastic cell invasiveness.