CEACAM1 Promotes Melanoma Cell Growth through Sox-2

AbstractThe prognostic value of the carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) in melanoma was demonstrated more than a decade ago as superior to Breslow score. We have previously shown that intercellular homophilic CEACAM1 interactions protect melanoma cells from lymphocyte-mediated elimination. Here, we study the direct effects of CEACAM1 on melanoma cell biology. By employing tissue microarrays and low-passage primary cultures of metastatic melanoma, we show that CEACAM1 expression gradually increases from nevi to metastatic specimens, with a strong dominance of the CEACAM1-Long tail splice variant. Using experimental systems of CEACAM1 knockdown and overexpression of selective variants or truncation mutants, we prove that only the full-length long tail variant enhances melanoma cell proliferation in vitro and in vivo. This effect is not reversed with a CEACAM1-blocking antibody, suggesting that it is not mediated by intercellular homophilic interactions. Downstream, CEACAM1-Long increases the expression of Sox-2, which we show to be responsible for the CEACAM1-mediated enhanced proliferation. Furthermore, analysis of the CEACAM1 promoter reveals two single-nucleotide polymorphisms (SNPs) that significantly enhance the promoter's activity compared with the consensus nucleotides. Importantly, case-control genetic SNP analysis of 134 patients with melanoma and matched healthy donors show that patients with melanoma do not exhibit the Hardy-Weinberg balance and that homozygous SNP genotype enhances the hazard ratio to develop melanoma by 35%. These observations shed new mechanistic light on the role of CEACAM1 in melanoma, forming the basis for development of novel therapeutic and diagnostic technologies

Outcome of Second Primary Malignancies Developing in Multiple Myeloma Patients

Background: There is an increased risk of second primary malignancies (SMPs) in patients with multiple myeloma (MM). This multinational 'real-world' retrospective study analyzed the characteristics and outcomes of MM patients that developed SPMs.Results: 165 patients were analyzed: 62.4% males; 8.5% with a prior cancer; 113 with solid SPMs, mainly =stage 2; and 52 with hematological SPM (hemato-SPM), mainly MDS/AML. Patients with hemato-SPM were younger (p = 0.05) and more frequently had a prior AutoHCT (p = 0.012). The time to SPM was shorter in the older (>65 years) and more heavily pretreated patients. One hundred patients were actively treated at the time of SPM detection. Treatment was discontinued in 52, substituted with another anti-MM therapy in 15, and continued in 33 patients. Treatment discontinuation was predominant in the patients diagnosed with hemato-SPM (76%). The median OS following SPM detection was 8.5 months, and the main cause of death was SPM. A poor ECOG status predicted a shorter OS (PS 3 vs. 0, HR = 5.74, 2.32-14.21, p < 0.001), whereas a normal hemoglobin level (HR = 0.43, 0.19-0.95, p = 0.037) predicted longer OS.Conclusions: With the continuing improvement in OS, a higher proportion of MM patients might develop SPM. The OS following SPM diagnosis is poor; hence, frequent surveillance and early detection are imperative to improve outcomes

Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy

Cellular distribution and dynamics of mitochondria are regulated by several motor proteins and a microtubule network. In neurons, mitochondrial trafficking is crucial because of high energy needs and calcium ion buffering along axons to synapses during neurotransmission. The trafficking kinesin proteins (TRAKs) are well characterized for their role in lysosomal and mitochondrial trafficking in cells, especially neurons. Using whole exome sequencing, we identified homozygous truncating variants in TRAK1 (NM_001042646:c.287-2A > C), in six lethal encephalopathic patients from three unrelated families. The pathogenic variant results in aberrant splicing and significantly reduced gene expression at the RNA and protein levels. In comparison with normal cells, TRAK1-deficient fibroblasts showed irregular mitochondrial distribution, altered mitochondrial motility, reduced mitochondrial membrane potential, and diminished mitochondrial respiration. This study confirms the role of TRAK1 in mitochondrial dynamics and constitutes the first report of this gene in association with a severe neurodevelopmental disorder

The transient receptor potential vanilloid 2 cation channel is abundant in macrophages accumulating at the peri-infarct zone and may enhance their migration capacity towards injured cardiomyocytes following myocardial infarction.

PURPOSE: A novel family of transient receptor potential (TRP) channels, that may hold a role in calcium homeostasis, has recently been described. By employing a GeneChip array analysis we have demonstrated a clear and specific upregulation of the TRP vanilloid 2 (TRPV2) mRNA in the left ventricles (LV) 3-5 days post-acute myocardial infarction (MI) compared to sham-operated controls, both in rats and in mice. We sought to characterize the cardiac cellular subpopulations in which TRPV2 is overexpressed upon acute MI. METHODS: Lewis rats underwent an acute MI by ligation of the left anterior descending artery or chest opening only (sham). The animals were terminated at various time points and an immunohistochemical (IHC) and immunofluorescent (IFC) staining of the LV sections as well as a flow cytometry analysis of LV-derived cells were carried out, using anti-TRPV2 and anti-monocyte/macrophage antibodies. Rat alveolar macrophage cells, NR8383, transiently transfected with TRPV2 siRNA were allowed to migrate towards hypoxic conditioned media of the rat cardiac myoblast line H9C2 using a trans-well migration assay. The macrophage cells migrating to the bottom side of the inserts were counted. RESULTS: The IHC and IFC staining as well as the flow cytometry data demonstrated a substantial expression of TRPV2 in infiltrating macrophages in the peri-infarct region 3-5 days post-acute MI. The in vitro migration assay data demonstrated that following inhibition of the TRPV2 channel, the number of migrating macrophages towards conditioned medium of hypoxic cardiomyocytes was significantly reduced. CONCLUSIONS: TRPV2 is highly expressed on the peri-infarct infiltrating macrophages and may play an important role in post-MI phagocytosis. Better characterization of this channel may pave the way for identifying a new target for modulating the dramatic post-MI immune reactions

MiRNA expression in psoriatic skin: reciprocal regulation of hsa-miR-99a and IGF-1R.

BACKGROUND: Psoriasis is a complex disease at the cellular, genomic and genetic levels. The role of microRNAs in skin development was shown in a keratinocyte-specific Dicer knockout mouse model. Considering that two main characteristics of psoriasis are keratinocytes hyperproliferation and abnormal skin differentiation, we hypothesized that aberrant microRNA expression contributes to the psoriatic phenotype. Here, we describe the differential expression of miRNAs in psoriatic involved and uninvolved skin as compared to normal skin, revealing an additional aspect of this complex disorder. METHODOLOGY/PRINCIPAL FINDINGS: Expression arrays were used to compare microRNA expression in normal skin versus psoriatic involved and uninvolved skin. Fourteen differentially expressed microRNAs were identified, including hsa-miR-99a, hsa-miR-150, hsa-miR-423 and hsa-miR-197. The expression of these microRNAs was reevaluated by qPCR. IGF-1R, which is involved in skin development and the pathogenesis of psoriasis, is a predicted target of hsa-miR-99a. In an in situ hybridization assay, we found that IGF-1R and miR-99a are reciprocally expressed in the epidermis. Using a reporter assay, we found that IGF-1R is targeted by hsa-miR-99a. Moreover, over expression of miR-99a in primary keratinocytes down-regulates the expression of the endogenous IGF-1R protein. Over expression of miR-99a also inhibits keratinocyte proliferation and increases Keratin 10 expression. These findings suggest that overexpression of hsa-miR-99a in keratinocytes drives them towards differentiation. In primary keratinocytes grown in high Ca(++), miR-99a expression increases over time. Finally, we found that IGF1 increases the expression of miR-99a. CONCLUSIONS/SIGNIFICANCE: We identified several microRNAs that are expressed differentially in normal and psoriatic skin. One of these miRNAs is miR-99a that regulates the expression of IGF-1R. Moreover, miR-99a seems to play a role in the differentiation of keratinocytes. We suggest that miR-99a is one of the regulators of the IGF-1R signaling pathway in keratinocytes. Activation of IGF1 signaling results in elevation of miR-99a which represses the expression of IGF-1R

Fold change of several TRP genes (MI versus SHAM, 3 days post procedure) obtained by Affymetrix GeneChip array performed on RNA samples isolated from the LV tissue of mice in an in vivo murine model for myocardial infarction.

<p>Fold change of several TRP genes (MI versus SHAM, 3 days post procedure) obtained by Affymetrix GeneChip array performed on RNA samples isolated from the LV tissue of mice in an in vivo murine model for myocardial infarction.</p

Fold change of several TRP genes (MI versus SHAM, 5 days post procedure) obtained by Affymetrix GeneChip array performed on RNA samples isolated from the LV tissue of rats in an in vivo model for an acute MI.

<p>Fold change of several TRP genes (MI versus SHAM, 5 days post procedure) obtained by Affymetrix GeneChip array performed on RNA samples isolated from the LV tissue of rats in an in vivo model for an acute MI.</p

Proposed scheme for the potential IGF-1/TRPV2 crosstalk.

<p>Shortly after myocardial infarction IGF-1 leads to TRPV2 trafficking to the cell membrane of cardiomyocytes through the autocrine signaling pathway as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105055#pone.0105055-Anversa1" target="_blank">[23]</a>. Paracrine effects of IGF-1 on peri-infarct macrophages may also take place as it has been reported that IGF-1 peptide becomes detectable several days post MI in macrophages infiltrating the repair zone <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105055#pone.0105055-Matthews1" target="_blank">[25]</a>. IGF-1 may thus enhance TRPV2 trafficking to the cell membrane of adjacent macrophages via paracrine effects.</p

Elevated protein levels of TRPV2 and IGF-1 following acute MI.

<p>(A.1) Surface levels of TRPV2 at various time points post MI compared to sham determined by flow cytometry analysis. Cells positive for TRPV2 expression are gated and the percentages are given (A.2). Total TRPV2 protein level three days post MI compared to sham analyzed by Western blot. Left panel of the membrane was blotted with TRPV2 antibody and the right panel was blotted with TRPV2 antibody pre-incubated with a TRPV2 peptide (1∶1 ratio). (B) IGF-1 expression level three days post MI, detected by Western blot analysis.</p

TRPV2 and IGF-1 are overexpressed after acute MI.

<p>Real time validation analysis of TRPV2 expression at different time points (3, 10 and 30 days post MI; n = 4/group) is given both quantitatively (A) by calculating the mRNA expression levels using the ΔΔCt method (**<i>p</i> = 0.01) and qualitatively (B) by viewing the PCR products in an agarose gel. (C) Real time analysis for IGF-1 expression in the same mRNA samples used for the real-time analysis of TRPV2 (*<i>p</i><0.05).</p