Identification of New Vulnerabilities in Conjunctival Melanoma Using Image-Based High Content Drug Screening.

Recent evidence suggests that numerous similarities exist between the genomic landscapes of both conjunctival and cutaneous melanoma. Since alterations of several components of the MAP kinases, PI3K/mTOR, and cell cycle pathways have been reported in conjunctival melanoma, we decided to assess the sensitivity of conjunctival melanoma to targeted inhibition mostly of kinase inhibitors. A high content drug screening assay based on automated fluorescence microscopy was performed in three conjunctival melanoma cell lines with different genomic backgrounds with 489 kinase inhibitors and 53 other inhibitors. IC50 and apoptosis induction were respectively assessed for 53 and 48 compounds. The genomic background influenced the response to MAK and PI3K/mTOR inhibition, more specifically cell lines with BRAF V600E mutations were more sensitive to BRAF/MEK inhibition, while CRMM2 bearing the NRASQ61L mutation was more sensitive to PI3k/mTOR inhibition. All cell lines demonstrated sensitivity to cell cycle inhibition, being more pronounced in CRMM2, especially with polo-like inhibitors. Our data also revealed new vulnerabilities to Hsp90 and Src inhibition. This study demonstrates that the genomic background partially influences the response to targeted therapy and uncovers a large panel of potential vulnerabilities in conjunctival melanoma that may expand available options for the management of this tumor.

5-Hydroxymethylcytosine Loss in Conjunctival Melanoma.

AIMS: Conjunctival and cutaneous melanoma partially share similar clinical and molecular backgrounds. As 5-hydroxymethylcytosine (5-hmC) loss has been demonstrated in cutaneous melanoma, we decided to assess if similar changes were occurring in conjunctival melanoma. METHODS: 5-methylcytosine (5-mC), 5-hmC and TET2 were respectively identified by immunohistochemistry and RNA ISH in 40 conjunctival nevi and 37 conjunctival melanomas. Clinicopathological correlations were established. RESULTS: 5-mC, TET2 and 5-hmC were respectively identified in 67.5%, 95% and 100% of conjunctival nevi and in 81.1%, 35.1% and 54% of conjunctival melanomas. A significant 5-hmC and TET2 loss was identified in conjunctival melanoma comparing to nevus, as well as a significant correlation between TET2 and 5-hmC expression. In the melanomas, 5-hmC expression was only significantly associated with local lymphatic invasion, but not with other clinicopathological parameters. There was a correlation between TET2 expression and the localization of the tumors. 5-mC expression was not associated with any clinicopathological parameters. CONCLUSIONS: We identified a significant 5-hmC loss in conjunctival melanoma similar to cutaneous melanoma. This loss may possibly be attributed to TET2 loss or IDH1 mutations. 5-hmC loss in conjunctival melanoma may help in the differential diagnosis between atypical conjunctival nevus and conjunctival melanoma.

Renal Programming by Transient Postnatal Overfeeding: The Role of Senescence Pathways.

BACKGROUND: Early nutrition influences the risk of chronic kidney diseases (CKDs) development in adulthood. Mechanisms underlying the early programming of altered renal function remain incompletely understood. This study aims at characterizing the role of cell senescence pathways in early programming of CKD after transient postnatal overfeeding. MATERIALS AND METHODS: Reduced litters of 3 mice pups and standard litters of 9 mice pups were obtained to induce overfed animals during lactation and control animals, respectively. Animals were sacrificed at 24 days (weaning) or at 7 months of life (adulthood). Body weight, blood pressure, kidney weight, and glomerular count were assessed in both groups. Senescence pathways were investigated using ß-Galactosidase staining and Western blotting of P16, P21, P53, P-Rb/Rb, and Sirtuin 1 (Sirt1) proteins. RESULTS: Early overfed animals had a higher body weight, a higher blood pressure at adulthood, and a higher glomerular number endowment compared to the control group. A higher ß-Galactosidase activity, a significant increase in P53 protein expression (p = 0.0045) and a significant decrease in P-Rb/Rb ratio (p = 0.02), were observed at weaning in animals who underwent early postnatal overfeeding. Protein expression of Sirt1, a protective factor against accelerated stress-induced senescence, was significantly decreased (p = 0.03) at weaning in early overfed animals. CONCLUSION: Early postnatal overfeeding by litter size reduction is associated with increased expression of factors involved in cellular senescence pathways, and decreased expression of Sirt 1 in the mouse kidney at weaning. These alterations may contribute to CKD programming after early postnatal overfeeding.

Transient postnatal overfeeding causes liver stress-induced premature senescence in adult mice.

Unbalanced nutrition early in life is increasingly recognized as an important factor in the development of chronic, non-communicable diseases at adulthood, including metabolic diseases. We aimed to determine whether transient postnatal overfeeding (OF) leads to liver stress-induced premature senescence (SIPS) of hepatocytes in association with liver structure and hepatic function alterations. Litters sizes of male C57BL/6 mice were adjusted to 9 pups (normal feeding, NF) or reduced to 3 pups during the lactation period to induce transient postnatal OF. Compared to the NF group, seven-month-old adult mice transiently overfed during the postnatal period were overweight and developed glucose intolerance and insulin resistance. Their livers showed microsteatosis and fibrosis, while hepatic insulin signaling and glucose transporter protein expressions were altered. Increased hepatic oxidative stress (OS) was observed, with increased superoxide anion production, glucose-6-phosphate dehydrogenase protein expression, oxidative DNA damage and decreased levels of antioxidant defense markers, such as superoxide dismutase and catalase proteins. Hepatocyte senescence was characterized by increased p21WAF, p53, Acp53, p16INK4a and decreased pRb/Rb and Sirtuin-1 (SIRT-1) protein expression levels. Transient postnatal OF induces liver OS at adulthood, associated with hepatocyte SIPS and alterations in liver structure and hepatic functions, which could be mediated by a SIRT-1 deficiency.

Aldehyde dehydrogenase activity plays a Key role in the aggressive phenotype of neuroblastoma.

BACKGROUND: The successful targeting of neuroblastoma (NB) by associating tumor-initiating cells (TICs) is a major challenge in the development of new therapeutic strategies. The subfamily of aldehyde dehydrogenases 1 (ALDH1) isoenzymes, which comprises ALDH1A1, ALDH1A2, and ALDH1A3, is involved in the synthesis of retinoic acid, and has been identified as functional stem cell markers in diverse cancers. By combining serial neurosphere passages with gene expression profiling, we have previously identified ALDH1A2 and ALDH1A3 as potential NB TICs markers in patient-derived xenograft tumors. In this study, we explored the involvement of ALDH1 isoenzymes and the related ALDH activity in NB aggressive properties. METHODS: ALDH activity and ALDH1A1/A2/A3 expression levels were measured using the ALDEFLUOR™ kit, and by real-time PCR, respectively. ALDH activity was inhibited using the specific ALDH inhibitor diethylaminobenzaldehyde (DEAB), and ALDH1A3 gene knock-out was generated through the CRISPR/Cas9 technology. RESULTS: We first confirmed the enrichment of ALDH1A2 and ALDH1A3 mRNA expression in NB cell lines and patient-derived xenograft tumors during neurosphere passages. We found that high ALDH1A1 expression was associated with less aggressive NB tumors and cell lines, and correlated with favorable prognostic factors. In contrast, we observed that ALDH1A3 was more widely expressed in NB cell lines and was associated with poor survival and high-risk prognostic factors. We also identified an important ALDH activity in various NB cell lines and patient-derived xenograft tumors. Specific inhibition of ALDH activity with diethylaminobenzaldehyde (DEAB) resulted in a strong reduction of NB cell clonogenicity, and TIC self-renewal potential, and partially enhanced NB cells sensitivity to 4-hydroxycyclophosphamide. Finally, the specific knock-out of ALDH1A3 via CRISPR/Cas9 gene editing reduced NB cell clonogenicity, and mediated a cell type-dependent inhibition of TIC self-renewal properties. CONCLUSIONS: Together our data uncover the participation of ALDH enzymatic activity in the aggressive properties and 4-hydroxycyclophosphamide resistance of NB, and show that the specific ALDH1A3 isoenzyme increases the aggressive capacities of a subset of NB cells.

Functional sphere profiling reveals the complexity of neuroblastoma tumor-initiating cell model.

Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically "profile" the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.

Complex molecular mechanisms cooperate to mediate histone deacetylase inhibitors anti-tumour activity in neuroblastoma cells.

BACKGROUND: Histone deacetylase inhibitors (HDACi) are a new class of promising anti-tumour agent inhibiting cell proliferation and survival in tumour cells with very low toxicity toward normal cells. Neuroblastoma (NB) is the second most common solid tumour in children still associated with poor outcome in higher stages and, thus NB strongly requires novel treatment modalities. RESULTS: We show here that the HDACi Sodium Butyrate (NaB), suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA) strongly reduce NB cells viability. The anti-tumour activity of these HDACi involved the induction of cell cycle arrest in the G2/M phase, followed by the activation of the intrinsic apoptotic pathway, via the activation of the caspases cascade. Moreover, HDACi mediated the activation of the pro-apoptotic proteins Bid and BimEL and the inactivation of the anti-apoptotic proteins XIAP, Bcl-xL, RIP and survivin, that further enhanced the apoptotic signal. Interestingly, the activity of these apoptosis regulators was modulated by several different mechanisms, either by caspases dependent proteolytic cleavage or by degradation via the proteasome pathway. In addition, HDACi strongly impaired the hypoxia-induced secretion of VEGF by NB cells. CONCLUSION: HDACi are therefore interesting new anti-tumour agents for targeting highly malignant tumours such as NB, as these agents display a strong toxicity toward aggressive NB cells and they may possibly reduce angiogenesis by decreasing VEGF production by NB cells.