NALCN-mediated sodium influx confers metastatic prostate cancer cell invasiveness.

There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na+ /Ca2+ signature required for persistent invasion. We identify the Na+ leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca2+ oscillations required for invadopodia formation. Indeed, NALCN-mediated Na+ influx into cancer cells maintains intracellular Ca2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na+ /Ca2+ exchangers, SERCA and store-operated channels. This signaling cascade promotes activity of the NACLN-colocalized proto-oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller.

Distinct Regulation of EZH2 and its Repressive H3K27me3 Mark in Polyomavirus-Positive and -Negative Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is an aggressive skin cancer for which Merkel cell polyomavirus integration and expression of viral oncogenes small T and Large T have been identified as major oncogenic determinants. Recently, a component of the PRC2 complex, the histone methyltransferase enhancer of zeste homolog 2 (EZH2) that induces H3K27 trimethylation as a repressive mark has been proposed as a potential therapeutic target in MCC. Because divergent results have been reported for the levels of EZH2 and trimethylation of lysine 27 on histone 3, we analyzed these factors in a large MCC cohort to identify the molecular determinants of EZH2 activity in MCC and to establish MCC cell lines' sensitivity to EZH2 inhibitors. Immunohistochemical expression of EZH2 was observed in 92% of MCC tumors (156 of 170), with higher expression levels in virus-positive than virus-negative tumors (P = 0.026). For the latter, we showed overexpression of EZHIP, a negative regulator of the PRC2 complex. In vitro, ectopic expression of the large T antigen in fibroblasts led to the induction of EZH2 expression, whereas the knockdown of T antigens in MCC cell lines resulted in decreased EZH2 expression. EZH2 inhibition led to selective cytotoxicity on virus-positive MCC cell lines. This study highlights the distinct mechanisms of EZH2 induction between virus-negative and -positive MCC.

Curcumin and NCLX inhibitors share anti-tumoral mechanisms in microsatellite-instability-driven colorectal cancer.

BACKGROUND AND AIMS: Recent evidences highlight a role of the mitochondria calcium homeostasis in the development of colorectal cancer (CRC). To overcome treatment resistance, we aimed to evaluate the role of the mitochondrial sodium-calcium-lithium exchanger (NCLX) and its targeting in CRC. We also identified curcumin as a new inhibitor of NCLX. METHODS: We examined whether curcumin and pharmacological compounds induced the inhibition of NCLX-mediated mitochondrial calcium (mtCa2+) extrusion, the role of redox metabolism in this process. We evaluated their anti-tumorigenic activity in vitro and in a xenograft mouse model. We analyzed NCLX expression and associations with survival in The Cancer Genome Atlas (TCGA) dataset and in tissue microarrays from 381 patients with microsatellite instability (MSI)-driven CRC. RESULTS: In vitro, curcumin exerted strong anti-tumoral activity through its action on NCLX with mtCa2+ and reactive oxygen species overload associated with a mitochondrial membrane depolarization, leading to reduced ATP production and apoptosis. NCLX inhibition with pharmacological and molecular approaches reproduced the effects of curcumin. NCLX inhibitors decreased CRC tumor growth in vivo. Both transcriptomic analysis of TCGA dataset and immunohistochemical analysis of tissue microarrays demonstrated that higher NCLX expression was associated with MSI status, and for the first time, NCLX expression was significantly associated with recurrence-free survival. CONCLUSIONS: Our findings highlight a novel anti-tumoral mechanism of curcumin through its action on NCLX and mitochondria calcium overload that could benefit for therapeutic schedule of patients with MSI CRC.

Pectoralis major muscle atrophy is associated with mitochondrial energy wasting in cachectic patients with gastrointestinal cancer.

BACKGROUND: Cancer cachexia is a multifactorial syndrome characterized by involuntary and pathological weight loss, mainly due to skeletal muscle wasting, resulting in a decrease in patients' quality of life, response to cancer treatments, and survival. Our objective was to investigate skeletal muscle alterations in cachectic cancer patients. METHODS: This is a prospective study of patients managed for pancreatic or colorectal cancer with an indication for systemic chemotherapy (METERMUCADIG - NCT02573974). One lumbar CT image was used to determine body composition. Patients were divided into three groups [8 noncachectic (NC), 18 with mild cachexia (MC), and 19 with severe cachexia (SC)] based on the severity of weight loss and muscle mass. For each patient, a pectoralis major muscle biopsy was collected at the time of implantable chamber placement. We used high-resolution oxygraphy to measure mitochondrial muscle oxygen consumption on permeabilized muscle fibres. We also performed optical and electron microscopy analyses, as well as gene and protein expression analyses. RESULTS: Forty-five patients were included. Patients were 67% male, aged 67 years (interquartile range, 59-77). Twenty-three (51%) and 22 (49%) patients were managed for pancreatic and colorectal cancer, respectively. Our results show a positive correlation between median myofibres area and skeletal muscle index (P = 0.0007). Cancer cachexia was associated with a decrease in MAFbx protein expression (P < 0.01), a marker of proteolysis through the ubiquitin-proteasome pathway. Mitochondrial oxygen consumption related to energy wasting was significantly increased (SC vs. NC, P = 0.028) and mitochondrial area tended to increase (SC vs. MC, P = 0.056) in SC patients. On the contrary, mitochondria content and networks remain unaltered in cachectic cancer patients. Finally, our results show no dysfunction in lipid storage and endoplasmic reticulum homeostasis. CONCLUSIONS: This clinical protocol brings unique data that provide new insight to mechanisms underlying muscle wasting in cancer cachexia. We report for the first time an increase in mitochondrial energy wasting in the skeletal muscle of severe cachectic cancer patients. Additional clinical studies are essential to further the exploring and understanding of these alterations.

Oncologic Impact and Safety of Pre-Operative Radiotherapy in Localized Prostate and Bladder Cancer: A Comprehensive Review from the Cancerology Committee of the Association Française d'Urologie.

Preoperative radiotherapy (RT) is commonly used for the treatment of various malignancies, including sarcomas, rectal, and gynaecological cancers, but it is preferentially used as a competitive treatment to radical surgery in uro-oncology or as a salvage procedure in cases of local recurrence. Nevertheless, preoperative RT represents an attractive strategy to prevent from intraoperative tumor seeding in the operative field, to sterilize microscopic extension outside the organ, and to enhance the pathological and/or imaging tumor response rate. Several clinical works support this research field in uro-oncology. In this review article, we summarized the oncologic impact and safety of preoperative RT in localized prostate and muscle-invasive bladder cancer. Preliminary studies suggest that both modalities can be complementary as initial primary tumor treatments and that a pre-operative radiotherapy strategy could be beneficial in a well-defined population of patients who are at a very high-risk of local relapse. Future prospective trials are warranted to evaluate the oncologic benefit of such a combination of local treatments in addition to new life-prolonging systemic therapies, such as immunotherapy, and new generation hormone therapies. Moreover, the safety and the feasibility of salvage surgical procedures due to non-response or local recurrence after pelvic RT remain poorly evaluated in that context.

Potential Targets Other Than PSMA for Prostate Cancer Theranostics: A Systematic Review.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is not sufficiently overexpressed in a small proportion of prostate cancer (PCa) patients, who require other strategies for imaging and/or treatment. We reviewed potential targets other than PSMA for PCa theranostics in nuclear medicine that have already been tested in humans. METHODS: We performed a systematic web search in the PubMed and Cochrane databases, with no time restrictions by pooling terms ("prostate cancer", "prostatic neoplasms") and ("radioligand", "radiotracer"). Included articles were clinical studies. The results were synthetized by the target type. RESULTS: We included 38 studies on six different targets: gastrin-releasing peptide receptors (GRPRs) (n = 23), androgen receptor (n = 11), somatostatin receptors (n = 6), urokinase plasminogen activator surface receptor (n = 4), fibroblast activation protein (n = 2 studies) and integrin receptors (n = 1). GRPRs, the most studied target, has a lower expression in high-grade PCa, CRPC and bone metastases. Its use might be of higher interest in treating earlier stages of PCa or low-grade PCa. Radiolabeled fibroblast activation protein inhibitors were the most recent and promising molecules, but specific studies reporting their interest in PCa are needed. CONCLUSION: Theranostics in nuclear medicine will continue to develop in the future, especially for PCa patients. Targets other than PSMA exist and deserve to be promoted.

The prognostic value of high-grade prostate cancer pattern on MRI-targeted biopsies: predictors for downgrading and importance of concomitant systematic biopsies.

PURPOSE: To assess the proportion and risk factors for downgrading and reclassification to favorable disease in patients having high-grade (HG) prostate cancer (PCa) pattern on magnetic resonance imaging (MRI)-targeted-biopsy (TB). METHODS: From a radical prostatectomy (RP) cohort, we included patients with pre-biopsy positive MRI and HG [defined by Grade Group (GG) ≥ 3] PCa on MRI-TB. All patients also underwent concomitant systematic biopsy (SB). The main endpoints were the rates of downgrading to GG2, overall downgrading, favorable disease (pT2 and GG2) on RP specimens, and biochemical recurrence-free-survival (RFS). We studied the correlations between HG on concomitant SB, final pathological outcomes and biochemical RFS curves. RESULTS: Overall downgrading, downgrading to GG2 disease and favorable disease were noted in 36.2%, 24.1%, and 15.4% respectively. HG on concomitant SB was correlated with pT3-4 disease (p < 0.001), pN1 disease (p < 0.001), positive surgical margins (p = 0.043), PSA recurrence (p = 0.003). In multivariable analysis, the presence of GG4-5 on TB (p = 0.013; OR 0.263) and the presence of HG on concomitant SB (p = 0.010; OR 0.269) were negatively and independently correlated with the risk of downgrading to GG2. The presence of HG on concomitant SB independently predicted RFS with a hazard ratio of 2.173 (p = 0.049; 95% CI 1.005-4.697). CONCLUSIONS: Our data shows that a limited HG restricted to TB can often be associated with a favorable grade in almost a quarter of the cases and downgraded in almost half of the cases. Detailed SB features, mainly the presence of HG on concomitant SB, was associated with a more accurate pathology and oncologic outcomes prediction, pleading for the maintenance of SB in MRI-positive patients.

Quantitative estimation of thrombus-erythrocytes using MRI. A phantom study with clot analogs and analysis by statistic regression models.

BACKGROUND: Thrombus composition has the potential to affect acute ischemic stroke (AIS) treatment. OBJECTIVE: To evaluate in an in vitro test the correlation of clot composition, especially erythrocytes (red blood cells (RBCs)), with the variation of signal intensity ratio (SIR) obtained with MRI sequences used for AIS, and qualification of the susceptibility vessel sign effect using clot analogs. MATERIALS AND METHODS: Nine ovine clots were fixed in a gelatin-manganese solution and studied by MRI (T2GE, T2-weighted gradient echo; SWI, susceptibility-weighted imaging; FLAIR, fluid attenuated inversion recovery). RBC concentration was estimated using regression models (SLR, single linear regression; MLR, multiple linear regression; RF, random Forest; and ANN, artificial neural networking), which combined the SIR-histology relationship of three MRI sequences. RESULTS: Negative correlation was found between SIR and RBC concentration. T2GE SWI could not statistically distinguish clots with RBC content >54% and <23%. SLR was applied only to FLAIR images since T2GE and SWI demonstrated signal saturation. All four regression models showed a correlation between MRI and histology: SLR=0.981; MLR=0.986; RF=0.994, and ANN=0.971. One unknown clot was studied and agreement between SIR and histological analyses was found in all models. CONCLUSIONS: We presented a method to quantify RBC concentration in clot analogs, combining SWI, T2GE, and FLAIR. This in vitro study has some limitations, so clot collection after thrombectomy with simultaneous imaging analysis is necessary to validate this model.

Enhancing Nab-Paclitaxel Delivery Using Microbubble-Assisted Ultrasound in a Pancreatic Cancer Model.

A combination of microbubbles (MBs) and ultrasound (US) is an emerging method for noninvasive and targeted enhancement of anti-cancer drug uptake. This method showed an increase local drug extravasation in tumor tissue while reducing the systemic adverse effects in various tumor models. The present study aims to evaluate the effectiveness of this approach for Nab-paclitaxel delivery in a pancreatic tumor model. US and MBs of different types in combination with Nab-paclitaxel showed a loss in cell viability of pancreatic cancer cells in comparison with Nab-paclitaxel treatment alone in in vitro scenario. The in vivo data revealed that US and MBs in combination with Nab-paclitaxel induced a significant decrease in the tumor volume in a subcutaneous pancreatic adenocarcinoma mouse model in comparison to tumors treated with Nab-paclitaxel alone. The postmortem anatomopathological analyses of tumor tissues partially confirmed these results. In conclusion, this study demonstrates that MB-assisted US is a relevant technology to increase the therapeutic effectiveness of Nab-paclitaxel in a pancreatic cancer model.

Contemporary assessment of the correlation between Bosniak classification and histological characteristics of surgically removed atypical renal cysts (UroCCR-12 study).

PURPOSE: To evaluate and compare pathological characteristics of renal cysts Bosniak IIF, III and IV in light of recent histological classification. PATIENTS AND METHODS: The French research network for kidney cancer UroCCR conducted a multicentre study on patients treated surgically for a renal cyst between 2007 and 2016. Independent radiological and centralized pathological reviews were performed for every patient. Pathological characteristics were compared to the Bosniak classification. RESULTS: Of a total 216 patients included, 175 (81.0%) tumours (90.9% of Bosniak IV, 69.8% of Bosniak III) were malignant or had a low malignant potential, with 60% of clear cell renal cell carcinoma (CCRCC), 24% of papillary RCC (PRCC) and 6.9% of multilocular cystic renal tumour of low malignant potential (MCRTLMP). Malignancies were mostly of low pT stage (86.4% of pT1-2), and low ISUP grade (68.0% of 1-2). Bosniak III cysts had a lower rate of CCRCC (46.7 vs. 67.3%), higher rate of PRCC (30 vs. 20.9%) and MCRTLMP (18.3 vs. 0.9%) compared to Bosniak IV (p < 0.001). Low-malignant potential lesions were less likely Bosniak IV and pT3-4 stage was more frequent in Bosniak IV vs. III (15.7 vs. 3.5%; p = 0.04). There were two recurrences (1.1%) and no cancer-related death occurred during follow-up. CONCLUSION: These results confirmed that cystic renal malignancies have excellent prognosis. Bosniak III cysts had a low malignant potential, which suggests surveillance could be an option for these lesions.

T-helper 1 immunoreaction influences survival in muscle-invasive bladder cancer: proof of concept.

OBJECTIVE: To define immunoscore in bladder cancer studying T helper 1 (Th1) immunoreaction. To define a cancer-specific survival model based on Th1 cells infiltration. METHODS: A total of 252 patients underwent primary transurethral resection of bladder tumour at our Institution. A retrospective review of a selected cohort with pT1 and muscle-invasive bladder cancer (MIBC) lesions was performed. Pathology blocks were marked with CD3 and CD8 antibodies. Immune cells density in stromal reaction (SR) was measured on five distinct high-power field (HPF) by two dedicated uro-pathologist blinded for patients' evolution. STATISTICS: Student test or non-parametric Wilcoxon test as appropriate to compare means between two groups. Receiver operating characteristics (ROC) curve to define markers threshold. Cox model to assess survival's predictors. RESULTS: Ten pT1 and 20 MIBC consecutive cases were analysed. Median follow-up was 33.4 months. Immunohistological analysis for pT1 lesions featured limited SR. For MIBC, the mean density of lymphocytes in the SR was of 105/HPF (CD3) and 86/HPF (CD8). Survivors harboured higher lymphocytes densities versus non survivors (CD3: p = 0.0319; CD8: p = 0.0279). CD3 (p = 0.034) and CD8 (p = 0.034) lymphocytes densities were independently associated with cancer-specific survival on Cox model analyses. The retrospective design and small size of cohorts are the study limitations. CONCLUSIONS: High CD3 and CD8 lymphocytes SR densities are associated with better cancer-specific survival for MIBC. Th1 reaction against the tumour seems to be protective for bladder cancer. Further evaluation is warranted.

Glial cell-derived neurotrophic factor expression in normal human lung and congenital cystic adenomatoid malformation.

CONTEXT: It has been recently suggested that dysregulation of developmental factors and disruption of cell turnover could play a role in the pathogenesis of congenital cystic adenomatoid malformation of the lung (CCAM). The glial cell-derived neurotrophic factor (GDNF) is a growth factor involved in organogenesis, and the temporal pattern of GDNF expression suggests that this factor may play a role in lung development. DESIGN: We studied GDNF expression by immunohistochemistry in postnatally resected CCAM of the lung (n = 10), normal fetal lung (n = 5), and normal postnatal lung (n = 5). We also studied the association between GDNF expression and both cell proliferation and apoptosis. RESULTS: GDNF was expressed in both epithelial and endothelial compartments of normal fetal lung, whereas no expression was found in normal postnatal lung. In contrast, in CCAM tissue, there was strong GDNF immunostaining that was restricted to epithelial cells. The percentage of proliferating epithelial cells was higher in CCAM tissue than in normal postnatal lung (6.3% vs 1.7%, P <.005). Apoptotic bodies were found in the mesenchyme of both normal fetal lung and CCAM tissue, whereas virtually no apoptotic bodies were detected in normal postnatal lung. CONCLUSIONS: Abnormal GDNF expression in CCAM suggests a dysregulation of the GDNF signaling pathway and argues in favor of a focal arrest in maturation during development. GDNF expression in lung tissue seems to be correlated with cell proliferation, suggesting that this factor could play a role in the growth of both fetal lung and CCAM.