CCN3 is a founding member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators. These secreted proteins are key regulators in embryonic development, and are associated with severe pathologies including fibrotic diseases and cancers. CCN3 was discovered as a MAV integration site in an avian nephroblastoma. Previous work established that the amino-truncated protein expressed in this tumor was inducing morphological transformation of chicken embryo fibroblasts, whereas the full-length secreted CCN3 protein was inhibiting cell growth. Amino-truncated variants were identified in cancer cell lines. Since the lack of signal peptide was expected to alter the fate of the truncated proteins, we hypothesized that modifications of CCN3 subcellular addressing could be responsible for the oncogenic activities of CCN3. The CCN proteins are composed of four structural modules (IGFBP, TSP1, VWC, and CT). We report that amino-truncated variants of CCN3 are addressed to the nucleus and that the carboxyterminal (CT) module of CCN3 is responsible for the nuclear addressing. Furthermore, our data identify nuclear CCN3 variants as potential transcriptional regulators. In this context, the CT module confers on nuclear CCN3 proteins a negative regulatory effect on transcription. We propose that the nuclear localization of amino-truncated CCN3 proteins be correlated to oncogenicity.

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BackgroundMortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312.ResultsThe aldehyde dehydrogenase (ALDH) positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect) and through decrease in telomere length (long-term effect). Administration of this telomerase inhibitor (40 mg/kg) in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls). Combination therapy consisting of irradiation (10Gy) plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length in vivo.ConclusionsWe conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer.

Previous work had suggested that recombinant CCN3 was partially inhibiting cell proliferation. Here we show that native CCN3 protein secreted into the conditioned medium of glioma transfected cells indeed induces a reduction in cell proliferation. Large amounts of CCN3 are shown to accumulate both cytoplasmically and extracellularly as cells reach high density, therefore highlighting new aspects on how cell growth may be regulated by CCN proteins. Evidence is presented establishing that the amount of CCN3 secreted into cell culture medium is regulated by post-translational proteolysis. As a consequence, the production of CCN3 varies throughout the cell cycle and CCN3 accumulates at the G2/M transition of the cycle. We also show that CCN3-induced inhibition of cell growth can be partially reversed by specific antibodies raised against a C-terminal peptide of CCN3. The use of several clones expressing various portions of CCN3 established that the CT module of CCN3 is sufficient to induce cell growth inhibition.

CCN proteins affect cell proliferation, migration, attachment, and differentiation. We identified CCN3 as a suppressed gene following platelet-derived growth factor (PDGF)-BB or -DD stimulation in a cDNA-array analysis of mesangial cells. In vitro growth-arrested mesangial cells overexpressed and secreted CCN3, whereas the addition of the recombinant protein inhibited cell growth. Induction of mesangial cell proliferation by PDGF-BB or the specific PDGF beta-receptor ligand PDGF-DD led to downregulation of CCN3 mRNA, confirming the array study. Specific PDGF alpha-receptor ligands had no effect. CCN3 protein was found in arterial smooth muscle cells, the medullary interstitium, and occasional podocytes in the healthy rat kidney. Glomerular CCN3 was low prior to mesangial proliferation but increased as glomerular cell proliferation subsided during mesangioproliferative glomerulonephritis (GN). Inhibition of PDGF-B in mesangioproliferative disease led to overexpression of glomerular CCN3 mRNA. CCN3 localized mostly to podocytes in human glomeruli, but this expression varied widely in different human glomerulonephritides. Glomerular cell proliferation negatively correlated with CCN3 expression in necrotizing GN. Our study identifies CCN3 as an endogenous inhibitor of mesangial cell growth and a modulator of PDGF-induced mitogenesis.

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Dry eye disease (DED) is characterized by an alteration of the tear film with ocular inflammation and neurosensory abnormalities. The main clinical signs of this condition are tear instability and ocular damage. Although DED has gained significant attention in the past few years, limited prescription treatment options are available for patients. Areas covered: The current manuscript summarizes the pre-clinical and clinical development of tivanisiran, a novel small interfering oligonucleotide of RNA (siRNA) used for the treatment of DED. Tivanisiran was designed to silence Transient Receptor Potential Vanilloid 1 (TRPV1); herein the chemistry and mechanism of action of this new compound is also described. Expert opinion: Drugs currently on the market mostly target the inflammatory component of the disease and show only partial efficacy. New compounds addressing other aspects of the disease would provide significant advantages and contribute to a more personalized treatment of the disease. Tivanisiran has been designed to reduce ocular discomfort and pain, and was shown to improve ocular hyperemia and tear quality in human and animal models. Consequently, if the results of the ongoing and future clinical trials meet their study endpoints, tivanisiran could be submitted to obtain approval for the treatment of DED.

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