ABSTRACT
During the tumorigenic process, cancer cells may become overly dependent on the activity of backup cellular pathways for their survival, representing vulnerabilities that could be exploited as therapeutic targets. Certain molecular vulnerabilities manifest as a synthetic lethality relationship, and the identification and characterization of new synthetic lethal interactions may pave the way for the development of new therapeutic approaches for human cancer. Our goal was to investigate a possible synthetic lethal interaction between a member of the Chromodomain Helicase DNA binding proteins family (CHD4) and a member of the histone methyltransferases family (SETDB1) in the molecular context of a cell line (Hs578T) representing the triple negative breast cancer (TNBC), a subtype of breast cancer lacking validated molecular targets for treatment. Therefore, we employed the CRISPR-Cas9 gene editing tool to individually or simultaneously introduce indels in the genomic loci corresponding to the catalytic domains of SETDB1 and CHD4 in the Hs578T cell line. Our main findings included: a) introduction of indels in exon 22 of SETDB1 sensitized Hs578T to the action of the genotoxic chemotherapy doxorubicin; b) by sequentially introducing indels in exon 22 of SETDB1 and exon 23 of CHD4 and tracking the percentage of the remaining wild-type sequences in the mixed cell populations generated, we obtained evidence of the existence of a synthetic lethality interaction between these genes. Considering the lack of molecular targets in TNBC, our findings provided valuable insights for development of new therapeutic approaches not only for TNBC but also for other cancer types.
ABSTRACT
Islet transplantation represents a therapeutic option for type 1 diabetes (T1D). Long-term viability of transplanted islets requires improvement. Mesenchymal stromal cells (MSCs) have been proposed as adjuvants for islet transplantation facilitating grafting and functionality. Stem cell aggregation provides physiological interactions between cells and enhances the in situ concentration of modulators of inflammation and immunity. We established a hanging-drop culture of adult human skin fibroblast-like cells as spheroids, and skin spheroid-derived cells (SphCs) were characterized. We assessed the potential of SphCs in improving islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model and characterized the secretome of SphCs by mass spectrometry-based proteomics. SphCs were characterized as multipotent progenitors and their coculture with anti-CD3 stimulated mouse splenocytes decreased CD4+ T cell proliferation with skewed cytokine secretion through an increase in the Th2/Th1 ratio profile. SphCs-conditioned media attenuated apoptosis of islets induced by cytokine challenge in vitro and importantly, intratesticular SphCs administration did not show tumorigenicity in immune-deficient mice. Moreover, SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in a diabetic mouse model without pharmacological immunosuppression. SphCs' protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influenced islets such as cytoprotection, cellular stress, metabolism, and survival. In summary, SphCs improved the performance of transplanted allogeneic islets in an experimental T1D model, without pharmacological immunosuppression. Future research is warranted to identify SphCs-secreted factors responsible for islets' endurance.
ABSTRACT
The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.
