Véronique Secq

A signature predictive of disease outcome in breast carcinomas, identified by quantitative immunocytochemical assays

Quantitative immunocytochemical assays of 1,200 breast carcinomas were assessed after construction of tissue microarrays. A total of 42 markers were evaluated for prognostic significance by univariate log rank test (mean follow‐up, 79 months), using quantitative scoring by an image analysis device and specific software. Complete data were obtained for 924 patients, for whom 27 of the 42 markers proved to be significant prognostic indicators. Analysis of these 27 markers by logistic regression showed that 18 (cMet, CD44v6, FAK, moesin, caveolin, c‐Kit, CK14, CD10, P21, P27, pMAPK, pSTAT3, STAT1, SHARP2, FYN, ER, PgR and c‐erb B2), and 15 when ER, PgR and c‐erb B2 were excluded, were 80.52% and 78.9% predictive of disease outcome, respectively. The immunocytochemical assays on 4 micron thick sections of fixed tissue are easy to handle in current practice and are cost‐effective. Quantitative densitometric measurement of immunoprecipitates by computer‐assisted devices from digitized microscopic images allows standardized high‐throughput “in situ” molecular profiling within tumors. It is concluded that this 15 marker immunohistochemical signature is suitable for current practice, since performed on paraffin sections of fixed tumor samples, and can be used to select patients needing more aggressive therapy, since this signature is about 80% predictive of poor clinical outcome. Also, the markers included in the signature may be indicative of tumor responsiveness to current chemotherapy or suggest new targets for specific therapies.

Cancer-associated fibroblast-derived annexin A6 + extracellular vesicles support pancreatic cancer aggressiveness

The intratumoral microenvironment, or stroma, is of major importance in the pathobiology of pancreatic ductal adenocarcinoma (PDA), and specific conditions in the stroma may promote increased cancer aggressiveness. We hypothesized that this heterogeneous and evolving compartment drastically influences tumor cell abilities, which in turn influences PDA aggressiveness through crosstalk that is mediated by extracellular vesicles (EVs). Here, we have analyzed the PDA proteomic stromal signature and identified a contribution of the annexin A6/LDL receptor-related protein 1/thrombospondin 1 (ANXA6/LRP1/TSP1) complex in tumor cell crosstalk. Formation of the ANXA6/LRP1/TSP1 complex was restricted to cancer-associated fibroblasts (CAFs) and required physiopathologic culture conditions that improved tumor cell survival and migration. Increased PDA aggressiveness was dependent on tumor cell-mediated uptake of CAF-derived ANXA6+ EVs carrying the ANXA6/LRP1/TSP1 complex. Depletion of ANXA6 in CAFs impaired complex formation and subsequently impaired PDA and metastasis occurrence, while injection of CAF-derived ANXA6+ EVs enhanced tumorigenesis. We found that the presence of ANXA6+ EVs in serum was restricted to PDA patients and represents a potential biomarker for PDA grade. These findings suggest that CAF-tumor cell crosstalk supported by ANXA6+ EVs is predictive of PDA aggressiveness, highlighting a therapeutic target and potential biomarker for PDA.

Homotypic cell cannibalism, a cell-death process regulated by the nuclear protein 1, opposes to metastasis in pancreatic cancer

Pancreatic adenocarcinoma (PDAC) is an extremely deadly disease for which all treatments available have failed to improve life expectancy significantly. This may be explained by the high metastatic potential of PDAC cells, which results from their dedifferentiation towards a mesenchymal phenotype. Some PDAC present cell‐in‐cell structures whose origin and significance are currently unknown. We show here that cell‐in‐cells form after homotypic cell cannibalism (HoCC). We found PDAC patients whose tumours display HoCC develop less metastasis than those without. In vitro, HoCC was promoted by inactivation of the nuclear protein 1 (Nupr1), and was enhanced by treatment with transforming growth factor β. HoCC ends with death of PDAC cells, consistent with a metastasis suppressor role for this phenomenon. Hence, our data indicates a protective role for HoCC in PDAC and identifies Nupr1 as a molecular regulator of this process.

Collagen-derived proline promotes pancreatic ductal adenocarcinoma cell survival under nutrient limited conditions

Tissue architecture contributes to pancreatic ductal adenocarcinoma (PDAC) phenotypes. Cancer cells within PDAC form gland-like structures embedded in a collagen-rich meshwork where nutrients and oxygen are scarce. Altered metabolism is needed for tumour cells to survive in this environment, but the metabolic modifications that allow PDAC cells to endure these conditions are incompletely understood. Here we demonstrate that collagen serves as a proline reservoir for PDAC cells to use as a nutrient source when other fuels are limited. We show PDAC cells are able to take up collagen fragments, which can promote PDAC cell survival under nutrient limited conditions, and that collagen-derived proline contributes to PDAC cell metabolism. Finally, we show that proline oxidase (PRODH1) is required for PDAC cell proliferation in vitro and in vivo. Collectively, our results indicate that PDAC extracellular matrix represents a nutrient reservoir for tumour cells highlighting the metabolic flexibility of this cancer.

Gene expression profiling of patient-derived pancreatic cancer xenografts predicts sensitivity to the BET bromodomain inhibitor JQ1: implications for individualized medicine efforts

c‐MYC controls more than 15% of genes responsible for proliferation, differentiation, and cellular metabolism in pancreatic as well as other cancers making this transcription factor a prime target for treating patients. The transcriptome of 55 patient‐derived xenografts show that 30% of them share an exacerbated expression profile of MYC transcriptional targets (MYC‐high). This cohort is characterized by a high level of Ki67 staining, a lower differentiation state, and a shorter survival time compared to the MYC‐low subgroup. To define classifier expression signature, we selected a group of 10 MYC target transcripts which expression is increased in the MYC‐high group and six transcripts increased in the MYC‐low group. We validated the ability of these markers panel to identify MYC‐high patient‐derived xenografts from both: discovery and validation cohorts as well as primary cell cultures from the same patients. We then showed that cells from MYC‐high patients are more sensitive to JQ1 treatment compared to MYC‐low cells, in monolayer, 3D cultured spheroids and in vivo xenografted tumors, due to cell cycle arrest followed by apoptosis. Therefore, these results provide new markers and potentially novel therapeutic modalities for distinct subgroups of pancreatic tumors and may find application to the future management of these patients within the setting of individualized medicine clinics.

Pancreatic Adenocarcinoma Therapeutic Targets Revealed by Tumor-Stroma Cross-Talk Analyses in Patient-Derived Xenografts

SUMMARY Preclinical models based on patient-derived xenografts have remarkable specificity in distinguishing transformed human tumor cells from non-transformed murine stromal cells computationally. We obtained 29 pancreatic ductal adenocarcinoma (PDAC) xenografts from either resectable or non-resectable patients (surgery and endoscopic ultrasound-guided fine-needle aspirate, respectively). Extensive multiomic profiling revealed two subtypes with distinct clinical outcomes. These subtypes uncovered specific alterations in DNA methylation and transcription as well as in signaling pathways involved in tumor-stromal cross-talk. The analysis of these pathways indicates therapeutic opportunities for targeting both compartments and their interactions. In particular, we show that inhibiting NPC1L1 with Ezetimibe, a clinically available drug, might be an efficient approach for treating pancreatic cancers. These findings uncover the complex and diverse interplay between PDAC tumors and the stroma and demonstrate the pivotal role of xenografts for drug discovery and relevance to PDAC.

A pancreatic ductal adenocarcinoma subpopulation is sensitive to FK866, an inhibitor of NAMPT

Treating pancreatic cancer is extremely challenging due to multiple factors, including chemoresistance and poor disease prognosis. Chemoresistance can be explained by: the presence of a dense stromal barrier leading to a lower vascularized condition, therefore limiting drug delivery; the huge intra-tumoral heterogeneity; and the status of epithelial-to-mesenchymal transition. These factors are highly variable between patients making it difficult to predict responses to chemotherapy. Nicotinamide phosphoribosyl transferase (NAMPT) is the main enzyme responsible for recycling cytosolic NAD+ in hypoxic conditions. FK866 is a noncompetitive specific inhibitor of NAMPT, which has proven anti-tumoral effects, although a clinical advantage has still not been demonstrated. Here, we tested the effect of FK866 on pancreatic cancer-derived primary cell cultures (PCCs), both alone and in combination with three different drugs typically used against this cancer: gemcitabine, 5-Fluorouracil (5FU) and oxaliplatin. The aims of this study were to evaluate the benefit of drug combinations, define groups of sensitivity, and identify a potential biomarker for predicting treatment sensitivity. We performed cell viability tests in the presence of either FK866 alone or in combination with the drugs above-mentioned. We confirmed both inter- and intra-tumoral heterogeneity. Interestingly, only the in vitro effect of gemcitabine was influenced by the addition of FK866. We also found that NAMPT mRNA expression levels can predict the sensitivity of cells to FK866. Overall, our results suggest that patients with tumors sensitive to FK866 can be identified using NAMPT mRNA levels as a biomarker and could therefore benefit from a co-treatment of gemcitabine plus FK866.

Distinct epigenetic landscapes underlie the pathobiology of pancreatic cancer subtypes

Recent studies have offered ample insight into genome-wide expression patterns to define pancreatic ductal adenocarcinoma (PDAC) subtypes, although there remains a lack of knowledge regarding the underlying epigenomics of PDAC. Here we perform multi-parametric integrative analyses of chromatin immunoprecipitation-sequencing (ChIP-seq) on multiple histone modifications, RNA-sequencing (RNA-seq), and DNA methylation to define epigenomic landscapes for PDAC subtypes, which can predict their relative aggressiveness and survival. Moreover, we describe the state of promoters, enhancers, super-enhancers, euchromatic, and heterochromatic regions for each subtype. Further analyses indicate that the distinct epigenomic landscapes are regulated by different membrane-to-nucleus pathways. Inactivation of a basal-specific super-enhancer associated pathway reveals the existence of plasticity between subtypes. Thus, our study provides new insight into the epigenetic landscapes associated with the heterogeneity of PDAC, thereby increasing our mechanistic understanding of this disease, as well as offering potential new markers and therapeutic targets.Pancreatic ductal adenocarcinoma (PDAC) is a complex disease and its underlying epigenomic heterogeneity is not fully understood. Here, the authors utilize patient-derived PDAC xenografts to define the epigenomic landscape of PDAC, highlighting chromatin states linked to differing disease aggressiveness and survival.

Novel role of VMP1 as modifier of the pancreatic tumor cell response to chemotherapeutic drugs.

We hypothesized that inhibiting molecules that mediate the adaptation response to cellular stress can antagonize the resistance of pancreatic cancer cells to chemotherapeutic drugs. Toward this end, here, we investigated how VMP1, a stress‐induced autophagy‐associated protein, modulate stress responses triggered by chemotherapeutic agents in PDAC. We find that VMP1 is particularly over‐expressed in poorly differentiated human pancreatic cancer. Pharmacological studies show that drugs that work, in part, via the endoplasmic reticulum stress response, induce VMP1 expression. Similarly, VMP1 is induced by known endoplasmic reticulum stress activators. Genetic inactivation of VMP1 using RNAi‐based antagonize the pancreatic cancer stress response to antitumoral agents. Functionally, we find that VMP1 regulates both autophagy and chemotherapeutic resistance even in the presence of chloroquin, ATG5 or Beclin 1 siRNAs, or a Beclin 1‐binding VMP1 mutant. In addition, VMP1 modulates endoplasmic reticulum stress independently of its coupling to the molecular and cellular autophagy machinery. Preclinical studies demonstrate that xenografts expressing an inducible and tractable form of VMP1 show increased resistance to the gemcitabine treatment. These results underscore a novel role for VMP1 as a potential therapeutic target for combinatorial therapies aimed at sensitizing pancreatic cancer cells to chemotherapeutic agents as well as provide novel molecular mechanisms to better understand this phenomenon. J. Cell. Physiol. 228: 1834–1843, 2013.

Pulmonary Endogenous Fluorescence Allows the Distinction of Primary Lung Cancer from the Perilesional Lung Parenchyma.

Background Pre-therapeutic pathological diagnosis is a crucial step of the management of pulmonary nodules suspected of being non small cell lung cancer (NSCLC), especially in the frame of currently implemented lung cancer screening programs in high-risk patients. Based on a human ex vivo model, we hypothesized that an embedded device measuring endogenous fluorescence would be able to distinguish pulmonary malignant lesions from the perilesional lung tissue. Methods Consecutive patients who underwent surgical resection of pulmonary lesions were included in this prospective and observational study over an 8-month period. Measurements were performed back table on surgical specimens in the operative room, both on suspicious lesions and the perilesional healthy parenchyma. Endogenous fluorescence signal was characterized according to three criteria: maximal intensity (Imax), wavelength, and shape of the signal (missing, stable, instable, photobleaching). Results Ninety-six patients with 111 suspicious lesions were included. Final pathological diagnoses were: primary lung cancers (n = 60), lung metastases of extra-thoracic malignancies (n = 27) and non-tumoral lesions (n = 24). Mean Imax was significantly higher in NSCLC targeted lesions when compared to the perilesional lung parenchyma (p<0,0001) or non-tumoral lesions (p<0,0001). Similarly, photobleaching was more frequently found in NSCLC than in perilesional lung (p<0,0001), or in non-tumoral lesions (p<0,001). Respective associated wavelengths were not statistically different between perilesional lung and either primary lung cancers or non-tumoral lesions. Considering lung metastases, both mean Imax and wavelength of the targeted lesions were not different from those of the perilesional lung tissue. In contrast, photobleaching was significantly more frequently observed in the targeted lesions than in the perilesional lung (p≤0,01). Conclusion Our results demonstrate that endogenous fluorescence applied to the diagnosis of lung nodules allows distinguishing NSCLC from the surrounding healthy parenchyma and from non-tumoral lesions. Inconclusive results were found for lung metastases due to the heterogeneity of this population.