A Novel Splicing Variant of COL2A1 in a Fetus with Achondrogenesis Type II: Interpretation of Pathogenicity of In-Frame Deletions.

Achondrogenesis type II (ACG2) is a lethal skeletal dysplasia caused by dominant pathogenic variants in COL2A1. Most of the variants found in patients with ACG2 affect the glycine residue included in the Gly-X-Y tripeptide repeat that characterizes the type II collagen helix. In this study, we reported a case of a novel splicing variant of COL2A1 in a fetus with ACG2. An NGS analysis of fetal DNA revealed a heterozygous variant c.1267-2_1269del located in intron 20/exon 21. The variant occurred de novo since it was not detected in DNA from the blood samples of parents. We generated an appropriate minigene construct to study the effect of the variant detected. The minigene expression resulted in the synthesis of a COL2A1 messenger RNA lacking exon 21, which generated a predicted in-frame deleted protein. Usually, in-frame deletion variants of COL2A1 cause a phenotype such as Kniest dysplasia, which is milder than ACG2. Therefore, we propose that the size and position of an in-frame deletion in COL2A1 may be relevant in determining the phenotype of skeletal dysplasia.

In Preclinical Model of Ovarian Cancer, the SGK1 Inhibitor SI113 Counteracts the Development of Paclitaxel Resistance and Restores Drug Sensitivity.

Ovarian cancer is the second most common gynecological malignancy worldwide. Paclitaxel is particularly important in the therapy of ovarian carcinomas, but the treatment efficacy is counteracted by the development of resistance to chemotherapy. The identification of target molecules that can prevent or control the development of chemoresistance might provide important tools for the management of patients affected by ovarian cancer. Serum- and glucocorticoid-regulated kinase 1 (SGK1) appears to be a key determinant of resistance to chemo- and radiotherapy. Specifically, SGK1 affects paclitaxel sensitivity in RKO colon carcinoma cells by modulating the specificity protein 1 (SP1)-dependent expression of Ran-specific GTPase-activating protein (RANBP1), a member of the GTP-binding nuclear protein Ran (RAN) network that is required for the organization and function of the mitotic spindle. SGK1 inhibition might thus be useful for counteracting the development of paclitaxel resistance. Here, we present in vitro data obtained using ovarian carcinoma cell lines that indicate that the SGK1 inhibitor SI113 inhibits cancer cell proliferation, potentiates the effects of paclitaxel-based chemotherapy, counteracts the development of paclitaxel resistance, and restores paclitaxel sensitivity in paclitaxel-resistant A2780 ovarian cancer cells. The results were corroborated by preclinical studies of xenografts generated in nude mice through the implantation of paclitaxel-resistant human ovarian cancer cells. The SGK1 inhibitor SI113 synergizes with paclitaxel in the treatment of xenografted ovarian cancer cells. Taken together, these data suggest that SGK1 inhibition should be investigated in clinical trials for the treatment of paclitaxel-resistant ovarian cancer.

Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells.

The fetal isoform A of the insulin receptor (IR-A) is frequently overexpressed in a variety of malignancies including breast cancer. IR overexpression has a recognized role in cancer progression and resistance to anticancer therapies. In particular, IR-A has a peculiar mitogenic potential and is activated not only by insulin but also by IGF-2. Previously, we identified discoidin domain receptor 1 (DDR1) as a new IR-A interacting protein. DDR1, a non-integrin collagen tyrosine kinase receptor, is overexpressed in several malignancies and plays a role in cancer progression and metastasis.We now evaluated whether DDR1 is able to exert a role in breast cancer biology by functionally cross-talking with IR. In MCF-7 human breast cancer cells, IR and DDR1 co-immunoprecipitated and co-localized after insulin or IGF-2 stimulation. In a panel of breast cancer cells, DDR1 knockdown by specific siRNAs markedly inhibited IR downstream signaling as well as proliferation, migration and colony formation in response to insulin and IGF-2. These effects were accompanied by reduction of IR protein and mRNA expression, which involved both transcriptional and post-transcriptional effects. DDR1 overexpression elicited opposite effects. Bioinformatics analysis of public domain databases showed that IR and DDR1 co-expression significantly correlates with several clinically relevant histopathological and molecular features of human breast carcinomas.These findings demonstrate that, in human breast cancer cells, DDR1 regulates IR expression and ligand dependent biological actions. This novel functional crosstalk is likely clinically relevant and may become a new molecular target in breast cancer.

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells.