Ketogenic HMG-CoA lyase and its product ß-hydroxybutyrate promote pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDA) tumor cells are deprived of oxygen and nutrients and therefore must adapt their metabolism to ensure proliferation. In some physiological states, cells rely on ketone bodies to satisfy their metabolic needs, especially during nutrient stress. Here, we show that PDA cells can activate ketone body metabolism and that ß-hydroxybutyrate (ßOHB) is an alternative cell-intrinsic or systemic fuel that can promote PDA growth and progression. PDA cells activate enzymes required for ketogenesis, utilizing various nutrients as carbon sources for ketone body formation. By assessing metabolic gene expression from spontaneously arising PDA tumors in mice, we find HMG-CoA lyase (HMGCL), involved in ketogenesis, to be among the most deregulated metabolic enzymes in PDA compared to normal pancreas. In vitro depletion of HMGCL impedes migration, tumor cell invasiveness, and anchorage-independent tumor sphere compaction. Moreover, disrupting HMGCL drastically decreases PDA tumor growth in vivo, while ßOHB stimulates metastatic dissemination to the liver. These findings suggest that ßOHB increases PDA aggressiveness and identify HMGCL and ketogenesis as metabolic targets for limiting PDA progression.

miR-424(322)/503 is a breast cancer tumor suppressor whose loss promotes resistance to chemotherapy.

The female mammary gland is a very dynamic organ that undergoes continuous tissue remodeling during adulthood. Although it is well established that the number of menstrual cycles and pregnancy (in this case transiently) increase the risk of breast cancer, the reasons are unclear. Growing clinical and experimental evidence indicates that improper involution plays a role in the development of this malignancy. Recently, we described the miR-424(322)/503 cluster as an important regulator of mammary epithelial involution after pregnancy. Here, through the analysis of ∼3000 primary tumors, we show that miR-424(322)/503 is commonly lost in a subset of aggressive breast cancers and describe the genetic aberrations that inactivate its expression. Furthermore, through the use of a knockout mouse model, we demonstrate for the first time that loss of miR-424(322)/503 promotes breast tumorigenesis in vivo. Remarkably, we found that loss of miR-424(322)/503 promotes chemoresistance due to the up-regulation of two of its targets: BCL-2 and insulin-like growth factor-1 receptor (IGF1R). Importantly, targeted therapies blocking the aberrant activity of these targets restore sensitivity to chemotherapy. Overall, our studies reveal miR-424(322)/503 as a tumor suppressor in breast cancer and provide a link between mammary epithelial involution, tumorigenesis, and the phenomenon of chemoresistance.

The serine/threonine kinase MINK1 directly regulates the function of promigratory proteins.

Upregulation of the developmental Wnt planar cell polarity (Wnt/PCP) pathway is observed in many cancers and is associated with cancer development. We have recently shown that PRICKLE1, a core Wnt/PCP pathway component, is a marker of poor prognosis in triple-negative breast cancer (TNBC). PRICKLE1 is phosphorylated by the serine/threonine kinase MINK1 and contributes to TNBC cell motility and invasiveness. However, the identity of the substrates of MINK1 and the role of MINK1 enzymatic activity in this process remain to be addressed. We used a phosphoproteomic strategy to identify MINK1 substrates, including LL5ß (also known as PHLDB2). LL5ß anchors microtubules at the cell cortex through its association with CLASP proteins to trigger focal adhesion disassembly. LL5ß is phosphorylated by MINK1, promoting its interaction with CLASP proteins. Using a kinase inhibitor, we demonstrate that the enzymatic activity of MINK1 is involved in PRICKLE1-LL5ß complex assembly and localization, as well as in cell migration. Analysis of gene expression data reveals that the concomitant upregulation of levels of mRNA encoding PRICKLE1 and LL5ß, which are MINK1 substrates, is associated with poor metastasis-free survival in TNBC patients. Taken together, our results suggest that MINK1 may represent a potential target for treatment of TNBC.

Mutational landscape of inflammatory breast cancer.

BACKGROUND: Inflammatory breast cancer (IBC) is the most pro-metastatic form of BC. Better understanding of its enigmatic pathophysiology is crucial. We report here the largest whole-exome sequencing (WES) study of clinical IBC samples. METHODS: We retrospectively applied WES to 54 untreated IBC primary tumor samples and matched normal DNA. The comparator samples were 102 stage-matched non-IBC samples from TCGA. We compared the somatic mutational profiles, spectra and signatures, copy number alterations (CNAs), HRD and heterogeneity scores, and frequencies of actionable genomic alterations (AGAs) between IBCs and non-IBCs. The comparisons were adjusted for the molecular subtypes. RESULTS: The number of somatic mutations, TMB, and mutational spectra were not different between IBCs and non-IBCs, and no gene was differentially mutated or showed differential frequency of CNAs. Among the COSMIC signatures, only the age-related signature was more frequent in non-IBCs than in IBCs. We also identified in IBCs two new mutational signatures not associated with any environmental exposure, one of them having been previously related to HIF pathway activation. Overall, the HRD score was not different between both groups, but was higher in TN IBCs than TN non-IBCs. IBCs were less frequently classified as heterogeneous according to heterogeneity H-index than non-IBCs (21% vs 33%), and clonal mutations were more frequent and subclonal mutations less frequent in IBCs. More than 50% of patients with IBC harbored at least one high-level of evidence (LOE) AGA (OncoKB LOE 1-2, ESCAT LOE I-II), similarly to patients with non-IBC. CONCLUSIONS: We provide the largest mutational landscape of IBC. Only a few subtle differences were identified with non-IBCs. The most clinically relevant one was the higher HRD score in TN IBCs than in TN non-IBCs, whereas the most intriguing one was the smaller intratumor heterogeneity of IBCs.

Whole-exome profiles of inflammatory breast cancer and pathological response to neoadjuvant chemotherapy.

BACKGROUND: Neoadjuvant chemotherapy (NACT) became a standard treatment strategy for patients with inflammatory breast cancer (IBC) because of high disease aggressiveness. However, given the heterogeneity of IBC, no molecular feature reliably predicts the response to chemotherapy. Whole-exome sequencing (WES) of clinical tumor samples provides an opportunity to identify genomic alterations associated with chemosensitivity. METHODS: We retrospectively applied WES to 44 untreated IBC primary tumor samples and matched normal DNA. The pathological response to NACT, assessed on operative specimen, distinguished the patients with versus without pathological complete response (pCR versus no-pCR respectively). We compared the mutational profiles, spectra and signatures, pathway mutations, copy number alterations (CNAs), HRD, and heterogeneity scores between pCR versus no-pCR patients. RESULTS: The TMB, HRD, and mutational spectra were not different between the complete (N = 13) versus non-complete (N = 31) responders. The two most frequently mutated genes were TP53 and PIK3CA. They were more frequently mutated in the complete responders, but the difference was not significant. Only two genes, NLRP3 and SLC9B1, were significantly more frequently mutated in the complete responders (23% vs. 0%). By contrast, several biological pathways involved in protein translation, PI3K pathway, and signal transduction showed significantly higher mutation frequency in the patients with pCR. We observed a higher abundance of COSMIC signature 7 (due to ultraviolet light exposure) in tumors from complete responders. The comparison of CNAs of the 3808 genes included in the GISTIC regions between both patients' groups identified 234 genes as differentially altered. The CIN signatures were not differentially represented between the complete versus non-complete responders. Based on the H-index, the patients with heterogeneous tumors displayed a lower pCR rate (11%) than those with less heterogeneous tumors (35%). CONCLUSIONS: This is the first study aiming at identifying correlations between the WES data of IBC samples and the achievement of pCR to NACT. Our results, obtained in this 44-sample series, suggest a few subtle genomic alterations associated with pathological response. Additional investigations are required in larger series.

Macrophages reprogramming driven by cancer-associated fibroblasts under FOLFIRINOX treatment correlates with shorter survival in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains a clinically challenging cancer, mainly due to limited therapeutic options and the presence of a highly prominent tumor microenvironment (TME), facilitating tumor progression. The TME is predominated by heterogeneous populations of cancer-associated fibroblasts (CAFs) and tumor associated macrophages (TAMs), in constant communication with each other and with tumor cells, influencing many tumoral abilities such as therapeutic resistance. However how the crosstalk between CAFs and macrophages evolves following chemotherapeutic treatment remains poorly understood, limiting our capacity to halt therapeutic resistance. METHODS: We combined biological characterization of macrophages indirectly cocultured with human PDAC CAFs, under FOLFIRINOX treatment, with mRNAseq analyses of such macrophages and evaluated the relevance of the specific gene expression signature in a large series of primary PDAC patients to search for correlation with overall survival (OS) after FOLFIRINOX chemotherapy. RESULTS: Firstly, we demonstrated that CAFs polarize naïve and M1 macrophages towards an M2-like phenotype with a specific increase of CD200R and CD209 M2 markers. Then, we demonstrated that CAFs counteract the pro-inflammatory phenotype induced by the FOLFIRINOX on Macrophages. Indeed, we highlighted that, under FOLFIRINOX, CAFs limit the FOLFIRINOX-induced cell death of macrophages and further reinforce their M2 phenotype as well as their immunosuppressive impact through specific chemokines production. Finally, we revealed that under FOLFIRINOX CAFs drive a specific macrophage gene expression signature involving SELENOP and GOS2 that correlates with shortened OS in FOLFIRINOX-treated PDAC patients. CONCLUSION: Our study provides insight into the complex interactions between TME cells under FOLFIRINOX treatment. It suggests potential novel candidates that could be used as therapeutic targets in combination with FOLFIRINOX to prevent and alleviate TME influx on therapeutic resistance as well as biomarkers to predict FOLFIRINOX response in PDAC patients. Video Abstract.

DDX5 mRNA-targeting antisense oligonucleotide as a new promising therapeutic in combating castration-resistant prostate cancer.

The heat shock protein 27 (Hsp27) has emerged as a principal factor of the castration-resistant prostate cancer (CRPC) progression. Also, an antisense oligonucleotide (ASO) against Hsp27 (OGX-427 or apatorsen) has been assessed in different clinical trials. Here, we illustrate that Hsp27 highly regulates the expression of the human DEAD-box protein 5 (DDX5), and we define DDX5 as a novel therapeutic target for CRPC treatment. DDX5 overexpression is strongly correlated with aggressive tumor features, notably with CRPC. DDX5 downregulation using a specific ASO-based inhibitor that acts on DDX5 mRNAs inhibits cell proliferation in preclinical models, and it particularly restores the treatment sensitivity of CRPC. Interestingly, through the identification and analysis of DDX5 protein interaction networks, we have identified some specific functions of DDX5 in CRPC that could contribute actively to tumor progression and therapeutic resistance. We first present the interactions of DDX5 and the Ku70/80 heterodimer and the transcription factor IIH, thereby uncovering DDX5 roles in different DNA repair pathways. Collectively, our study highlights critical functions of DDX5 contributing to CRPC progression and provides preclinical proof of concept that a combination of ASO-directed DDX5 inhibition with a DNA damage-inducing therapy can serve as a highly potential novel strategy to treat CRPC.

SK2 channels set a signalling hub bolstering CAF-triggered tumourigenic processes in pancreatic cancer.

OBJECTIVE: Intercellular communication within pancreatic ductal adenocarcinoma (PDAC) dramatically contributes to metastatic processes. The underlying mechanisms are poorly understood, resulting in a lack of targeted therapy to counteract stromal-induced cancer cell aggressiveness. Here, we investigated whether ion channels, which remain understudied in cancer biology, contribute to intercellular communication in PDAC. DESIGN: We evaluated the effects of conditioned media from patient-derived cancer-associated fibroblasts (CAFs) on electrical features of pancreatic cancer cells (PCC). The molecular mechanisms were deciphered using a combination of electrophysiology, bioinformatics, molecular and biochemistry techniques in cell lines and human samples. An orthotropic mouse model where CAF and PCC were co-injected was used to evaluate tumour growth and metastasis dissemination. Pharmacological studies were carried out in the Pdx1-Cre, Ink4afl/fl LSL-KrasG12D (KICpdx1) mouse model. RESULTS: We report that the K+ channel SK2 expressed in PCC is stimulated by CAF-secreted cues (8.84 vs 2.49 pA/pF) promoting the phosphorylation of the channel through an integrin-epidermal growth factor receptor (EGFR)-AKT (Protein kinase B) axis. SK2 stimulation sets a positive feedback on the signalling pathway, increasing invasiveness in vitro (threefold) and metastasis formation in vivo. The CAF-dependent formation of the signalling hub associating SK2 and AKT requires the sigma-1 receptor chaperone. The pharmacological targeting of Sig-1R abolished CAF-induced activation of SK2, reduced tumour progression and extended the overall survival in mice (11.7 weeks vs 9.5 weeks). CONCLUSION: We establish a new paradigm in which an ion channel shifts the activation level of a signalling pathway in response to stromal cues, opening a new therapeutic window targeting the formation of ion channel-dependent signalling hubs.

Immunologic constant of rejection as a predictive biomarker of immune checkpoint inhibitors efficacy in non-small cell lung cancer.

BACKGROUND: Anti-PD1/PDL1 immune checkpoint inhibitors (ICI) transformed the prognosis of patients with advanced non-small cell lung cancer (NSCLC). However, the response rate remains disappointing and toxicity may be life-threatening, making urgent identification of biomarkers predictive for efficacy. Immunologic Constant of Rejection signature (ICR) is a 20-gene expression signature of cytotoxic immune response with prognostic value in some solid cancers. Our objective was to assess its predictive value for benefit from anti-PD1/PDL1 in patients with advanced NSCLC. METHODS: We retrospectively profiled 44 primary tumors derived from NSCLC patients treated with ICI as single-agent in at least the second-line metastatic setting. Transcriptomic analysis was performed using the nCounter® analysis system and the PanCancer Immune Profiling Panel. We then pooled our data with clinico-biological data from four public gene expression data sets, leading to a total of 162 NSCLC patients treated with single-agent anti-PD1/PDL1. ICR was applied to all samples and correlation was searched between ICR classes and the Durable Clinical Benefit (DCB), defined as stable disease or objective response according to RECIST 1.1 for a minimum of 6 months after the start of ICI. RESULTS: The DCB rate was 29%; 22% of samples were classified as ICR1, 30% ICR2, 22% ICR3, and 26% ICR4. These classes were not associated with the clinico-pathological variables, but showed enrichment from ICR1 to ICR4 in quantitative/qualitative markers of immune response. ICR2-4 class was associated with a 5.65-fold DCB rate when compared with ICR1 class. In multivariate analysis, ICR classification remained associated with DCB, independently from PDL1 expression and other predictive immune signatures. By contrast, it was not associated with disease-free survival in 556 NSCLC TCGA patients untreated with ICI. CONCLUSION: The 20-gene ICR signature was independently associated with benefit from anti-PD1/PDL1 ICI in patients with advanced NSCLC. Validation in larger retrospective and prospective series is warranted.

Clinical, pathological, and comprehensive molecular analysis of the uterine clear cell carcinoma: a retrospective national study from TMRG and GINECO network.

BACKGROUND: Uterine clear cell carcinomas (CCC) represent less than 5% of uterine cancers. Their biological characteristics and clinical management remain uncertain. A multicenter study to explore both clinical and molecular features of these rare tumors was conducted. METHODS: This multicenter retrospective national study was performed within the French TMRG (Rare Gynecologic Malignant Tumors) network. Clinical data and, when available, FFPE blocks were collected. Clinical features, treatments, and outcome (progression-free survival (PFS) and overall survival (OS)) were analyzed and correlated to the protein (tissue micro-array), RNA (Nanostring nCounter® technology), and DNA (array-Comparative Genomic hybridization and target-next generation sequencing) levels using the tumor samples available. RESULTS: Sixty-eight patients with uterine CCC were enrolled, 61 from endometrial localization and 5 with cervix localization. Median age at diagnosis was 68.9 years old (range 19-89.7). Most tumors were diagnosed at an early stage (78% FIGO stage I-II). Hysterectomy (performed in 90%) and lymph node dissection (80%) were the most frequent surgical treatment. More than 70% of patients received external beam radiotherapy and 57% received brachytherapy. Nearly half (46%) of the patients received chemotherapy. After a median follow-up of 24.7 months, median PFS was 64.8 months (95 CI [5.3-124.4]) and median OS was 79.7 (IC95 [31.0-128.4]). Low hormone receptor expression (13% estrogen-receptor positive), frequent PI3K pathway alterations (58% PTEN loss, 50% PIK3CA mutations), and P53 abnormalities (41%) were observed. Mismatch repair deficiency was identified in 20%. P16 expression was associated with shorter PFS (HR = 5.88, 95 CI [1.56-25], p = 0.009). Transcriptomic analyzes revealed a specific transcriptomic profile notably with a high expression of immune response-associated genes in uterine CCC displaying a very good overall prognosis. CONCLUSIONS: Uterine CCC reported to be potentially MSI high, hormone receptors negative, and sometimes TP53 mutated. However, some patients with immune response-associated features and better prognosis may be candidate to treatment de-escalation and immunotherapy.

CSPG4 expression in soft tissue sarcomas is associated with poor prognosis and low cytotoxic immune response.

BACKGROUND: Soft tissue sarcomas (STS) are heterogeneous and pro-metastatic tumors. Identification of accurate prognostic factors and novel therapeutic targets are crucial. CSPG4 is a cell surface proteoglycan with oncogenic functions. It recently emerged as a potential target for immunotherapy, including cell therapy based on CSPG4-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CSPG4-CAR.CIKs) in STS. However, expression of CSPG4 is poorly known in STS so far. METHODS: We analyzed CSPG4 gene expression in 1378 localized STS clinical samples, and searched for correlations with clinicopathological data, including disease-free survival (DFS), and with tumor immune features. RESULTS: CSPG4 expression was heterogeneous across samples. High expression was associated with younger patients' age, more frequent undifferentiated pleomorphic sarcoma and myxofibrosarcoma pathological subtypes, more frequent internal trunk tumor site, and more CINSARC high-risk samples. No correlation existed with pathological tumor size and grade, and tumor depth. Patients with high CSPG4 expression displayed 49% (95% CI 42-57) 5-year DFS versus 61% (95% CI 56-68) in patients with low expression (p = 3.17E-03), representing a 49% increased risk of event in the "CSPG4-high" group (HR = 1.49, 95% CI 1.14-1.94). This unfavorable prognostic value persisted in multivariate analysis, independently from other variables. There were significant differences in immune variables between "CSPG4-high" and "CSPG4-low" tumors. The "CSPG4-low" tumors displayed profiles suggesting higher anti-tumor cytotoxic immune response and higher potential vulnerability to immune checkpoint inhibitors (ICI). By contrast, the "CSPG4-high" tumors displayed profiles implying an immune-excluded tumor microenvironment, potentially induced by hypoxia, resulting from an immature chaotic microvasculature, and/or the presence of contractile myofibroblasts. CONCLUSIONS: Patients with "CSPG4-high" STS, theoretically candidate for CAR.CIKs, display shorter DFS and an immune environment unfavorable to vulnerability to CAR.CIKs, which could be improved by combining anti-angiogenic drugs able to normalize the tumor vasculature. By contrast, "CSPG4-low" STS are better candidates for immune therapy involving ICI.

Molecular classification as prognostic factor and guide for treatment decision of pancreatic cancer.

Clinico-pathological factors fail to consistently predict the outcome after pancreatic resection for pancreatic ductal adenocarcinoma (PDAC). PDACs show a high level of inter- and intra- tumor genetic heterogeneity. A molecular classification should help sort patients into less heterogeneous and more appropriate groups regarding the metastatic risk and the therapeutic response, with the consequences of better predicting evolution and better orienting the treatment. PDAC can be classified based on mutational subtypes and 18gene alterations. Whole-genome sequencing identified mutational signatures, mutational burden and hyper-mutated tumors with specific DNA repair defects. Their overlap/similarities allow the definition of molecular subtypes. DNA and RNA classifications can be used in prognosis assessment. They are useful in therapeutic choice for they allow the design of approaches that can predict the respective drug sensitivity of each molecular subtype. This review provides a comprehensive analysis of available molecular classifications in PDAC and how this can help guide clinical decisions.

Inflammatory breast cancer cells are characterized by abrogated TGFß1-dependent cell motility and SMAD3 activity.

PURPOSE: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with elevated metastatic potential, characterized by tumor emboli in dermal and parenchymal lymph vessels. This study has investigated the hypothesis that TGFß signaling is implicated in the molecular biology of IBC. METHODS: TGFß1-induced cell motility and gene expression patterns were investigated in three IBC and three non-IBC (nIBC) cell lines. Tissue samples from IBC and nIBC patients were investigated for the expression of nuclear SMAD2, SMAD3, and SMAD4. SMAD protein levels were related to gene expression data. RESULTS: TGFß1-induced cell motility was strongly abrogated in IBC cells (P = 0.003). Genes differentially expressed between IBC and nIBC cells post TGFß1 exposure revealed attenuated expression of SMAD3 transcriptional regulators, but overexpression of MYC target genes in IBC. IBC patient samples demonstrated a near absence of SMAD3 and -4 expression in the primary tumor compared to nIBC patient samples (P < 0.001) and a further reduction of staining intensity in tumor emboli. Integration of gene and protein expression data revealed that a substantial fraction of the IBC signature genes correlated with SMAD3 and these genes are indicative of attenuated SMAD3 signaling in IBC. CONCLUSION: We demonstrate attenuated SMAD3 transcriptional activity and SMAD protein expression in IBC, together with obliterated TGFß1-induced IBC cell motility. The further reduction of nuclear SMAD expression levels in tumor emboli suggests that the activity of these transcription factors is involved in the metastatic dissemination of IBC cells, possibly by enabling collective invasion after partial EMT.

ECT2 associated to PRICKLE1 are poor-prognosis markers in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancers (TNBC) are poor-prognosis tumours candidate to chemotherapy as only systemic treatment. We previously found that PRICKLE1, a prometastatic protein involved in planar cell polarity, is upregulated in TNBC. We investigated the protein complex associated with PRICKLE1 in TNBC to identify proteins possibly involved in metastatic dissemination, which might provide new prognostic and/or therapeutic targets. METHODS: We used a proteomic approach to identify protein complexes associated with PRICKLE1. The mRNA expression levels of the corresponding genes were assessed in 8982 patients with invasive primary breast cancer. We then characterised the molecular interaction between PRICKLE1 and the guanine nucleotide exchange factor ECT2. Finally, experiments in Xenopus were carried out to determine their evolutionarily conserved interaction. RESULTS: Among the PRICKLE1 proteins network, we identified several small G-protein regulators. Combined analysis of the expression of PRICKLE1 and small G-protein regulators had a strong prognostic value in TNBC. Notably, the combined expression of ECT2 and PRICKLE1 provided a worst prognosis than PRICKLE1 expression alone in TNBC. PRICKLE1 regulated ECT2 activity and this interaction was evolutionary conserved. CONCLUSIONS: This work supports the idea that an evolutionarily conserved signalling pathway required for embryogenesis and activated in cancer may represent a suitable therapeutic target.

The immunologic constant of rejection classification refines the prognostic value of conventional prognostic signatures in breast cancer.

BACKGROUND: The immunologic constant of rejection (ICR) is a broad phenomenon of Th-1 immunity-mediated, tissue-specific destruction. METHODS: We tested the prognostic value of a 20-gene ICR expression signature in 8766 early breast cancers. RESULTS: Thirty-three percent of tumours were ICR1, 29% ICR2, 23% ICR3, and 15% ICR4. In univariate analysis, ICR4 was associated with a 36% reduction in risk of metastatic relapse when compared with ICR1-3 (p = 2.30E-03). In multivariate analysis including notably the three major prognostic signatures (Recurrence score, 70-gene signature, ROR-P), ICR was the strongest predictive variable (p = 9.80E-04). ICR showed no prognostic value in the HR+/HER2- subtype, but prognostic value in the HER2+ and TN subtypes. Furthermore, in each molecular subtype and among the tumours defined as high risk by the three prognostic signatures, ICR4 patients had a 41-75% reduction in risk of relapse as compared with ICR1-3 patients. ICR added significant prognostic information to that provided by the clinico-genomic models in the overall population and in each molecular subtype. ICR4 was independently associated with achievement of pathological complete response to neoadjuvant chemotherapy (p = 2.97E-04). CONCLUSION: ICR signature adds prognostic information to that of current proliferation-based signatures, with which it could be integrated to improve patients' stratification and guide adjuvant treatment.

PLZF mutation alters mouse hematopoietic stem cell function and cell cycle progression.

Hematopoietic stem cells (HSCs) give rise to all blood populations due to their long-term self-renewal and multipotent differentiation capacities. Because they have to persist throughout an organism's life span, HSCs tightly regulate the balance between proliferation and quiescence. Here, we investigated the role of the transcription factor promyelocytic leukemia zinc finger (plzf) in HSC fate using the Zbtb16(lu/lu)mouse model, which harbors a natural spontaneous mutation that inactivates plzf. Regenerative stress revealed that Zbtb16(lu/lu)HSCs had a lineage-skewing potential from lymphopoiesis toward myelopoiesis, an increase in the long-term-HSC pool, and a decreased repopulation potential. Furthermore, oldplzf-mutant HSCs present an amplified aging phenotype, suggesting that plzf controls age-related pathway. We found that Zbtb16(lu/lu)HSCs harbor a transcriptional signature associated with a loss of stemness and cell cycle deregulation. Lastly, cell cycle analyses revealed an important role for plzf in the regulation of the G1-S transition of HSCs. Our study reveals a new role for plzf in regulating HSC function that is linked to cell cycle regulation, and positions plzf as a key player in controlling HSC homeostasis.

Validation and comparison of the molecular classifications of pancreatic carcinomas.

Four molecular classifications of pancreatic ductal adenocarcinoma (PDAC), biologically and clinically relevant and based on gene expression profiles, were established in the recent years, including the Collisson's, Moffitt's ("tumor" and "stroma" classifications), and Bailey's classifications. The aim of this study was to validate the prognostic value of the Moffitt's classifications and to compare the Collisson's, Moffitt's, and Bailey's classifications in a large series of samples. We collected clinical and gene expression data of PDAC samples from 15 public data sets, resulting in a total of 846 primary cancer samples, including 601 with survival annotation. All samples were classified according to each of the four multigene classifiers. We confirmed the independent prognostic value of the Moffitt "tumor", Moffitt "stroma", and Bailey's classifications, but not that of the Collisson's classification. Despite a relatively low gene overlap, all classifications were associated with pathological grade, an important prognostic feature and reflect of intrinsic molecular characteristics of tumors. The concordance rate in term of "good-prognosis" vs. "poor-prognosis" prediction by classifiers was relatively high (from 73 to 86%) between the three "tumor" classifications based on tumor gene lists (Collisson, Moffitt "tumor", Bailey), but low (from 50 to 60%) with the Moffitt's stroma classification based on stroma genes. Multivariate analysis incorporating the four classifiers together retained as significant variables the Moffitt "stroma" and Bailey classifications, highlighting the complementarity of classifiers based on tumor epithelium (Bailey) and tumor stroma (Moffitt stroma). Our results reinforce the clinical validity of subtyping in PDAC, which should be regarded as a collection of separate diseases. Beside their clinical utility that remains to be demonstrated, the clinical interest of the subtypes, notably those from Bailey's and Moffitt's "stroma" classifiers that show independent prognostic value, will be reinforced by the identification of new biomarkers and/or therapeutic targets in each subtype for designing and testing novel specific targeted therapies.

A 25-gene classifier predicts overall survival in resectable pancreatic cancer.

BACKGROUND: Pancreatic carcinoma is one of the most lethal human cancers. In patients with resectable tumors, surgery followed by adjuvant chemotherapy is the only curative treatment. However, the 5-year survival is 20%. Because of a strong metastatic propensity, neoadjuvant chemotherapy is being tested in randomized clinical trials. In this context, improving the selection of patients for immediate surgery or neoadjuvant chemotherapy is crucial, and high-throughput molecular analyses may help; the present study aims to address this. METHODS: Clinicopathological and gene expression data of 695 pancreatic carcinoma samples were collected from nine datasets and supervised analysis was applied to search for a gene expression signature predictive for overall survival (OS) in the 601 informative operated patients. The signature was identified in a learning set of patients and tested for its robustness in a large independent validation set. RESULTS: Supervised analysis identified 1400 genes differentially expressed between two selected patient groups in the learning set, namely 17 long-term survivors (LTS; ≥ 36 months after surgery) and 22 short-term survivors (STS; dead of disease between 2 and 6 months after surgery). From these, a 25-gene prognostic classifier was developed, which identified two classes ("STS-like" and "LTS-like") in the independent validation set (n = 562), with a 25% (95% CI 18-33) and 48% (95% CI 42-54) 2-year OS (P = 4.33 × 10-9), respectively. Importantly, the prognostic value of this classifier was independent from both clinicopathological prognostic features and molecular subtypes in multivariate analysis, and existed in each of the nine datasets separately. The generation of 100,000 random gene signatures by a resampling scheme showed the non-random nature of our prognostic classifier. CONCLUSION: This study, the largest prognostic study of gene expression profiles in pancreatic carcinoma, reports a 25-gene signature associated with post-operative OS independently of classical factors and molecular subtypes. This classifier may help select patients with resectable disease for either immediate surgery (the LTS-like class) or neoadjuvant chemotherapy (the STS-like class). Its assessment in the current prospective trials of adjuvant and neoadjuvant chemotherapy trials is warranted, as well as the functional analysis of the classifier genes, which may provide new therapeutic targets.

Stromal Expression of MARCKS Protein in Ovarian Carcinomas Has Unfavorable Prognostic Value.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Identification of new therapeutic targets is crucial. MARCKS, myristoylated alanine-rich C-kinase substrate, has been implicated in aggressiveness of several cancers and MARCKS inhibitors are in development. Using immunohistochemistry (IHC), we retrospectively assessed MARCKS expression in epithelial and stromal cells of 118 pre-chemotherapy EOC samples and 40 normal ovarian samples from patients treated at Salah Azaiez Institute. We compared MARCKS expression in normal versus cancer samples, and searched for correlations with clinicopathological features, including overall survival (OS). Seventy-five percent of normal samples showed positive epithelial MARCKS staining versus 50% of tumor samples (p = 6.02 × 10-3). By contrast, stromal MARCKS expression was more frequent in tumor samples (77%) than in normal samples (22%; p = 1.41 × 10-9). There was no correlation between epithelial and stromal IHC MARCKS statutes and prognostic clinicopathological features. Stromal MARCKS expression was correlated with shorter poor OS in uni- and multivariate analyses. Stromal MARCKS overexpression in tumors might contribute to cancer-associated fibroblasts activation and to the poor prognosis of EOC, suggesting a potential therapeutic interest of MARCKS inhibition for targeting the cooperative tumor stroma.