A unique STK4 mutation truncating only the C-terminal SARAH domain results in a mild clinical phenotype despite severe T cell lymphopenia: Case report

Mutations in STK4 (MST1) are implicated in a form of autosomal recessive combined immunodeficiency, resulting in recurrent infections (especially Epstein-Barr virus viremia), autoimmunity, and cardiac malformations. Here we report a patient with an atypically mild presentation of this disease, initially presenting with severe T cell lymphopenia (< 500 per mm3) and intermittent neutropenia, but now surviving well on immunoglobulins and prophylactic antibacterial treatment. She harbors a unique STK4 mutation that lies further downstream than all others reported to date. Unlike other published cases, her mRNA transcript is not vulnerable to nonsense mediated decay (NMD) and yields a truncated protein that is expected to lose only the C-terminal SARAH domain. This domain is critical for autodimerization and autophosphorylation. While exhibiting significant differences from controls, this patient’s T cell proliferation defects and susceptibility to apoptosis are not as severe as reported elsewhere. Expression of PD-1 is in line with healthy controls. Similarly, the dysregulation seen in immunophenotyping is not as pronounced as in other published cases. The nature of this mutation, enabling its evasion from NMD, provides a rare glimpse into the clinical and cellular features associated with the absence of a “null” phenotype of this protein

Fascin Is a Key Regulator of Breast Cancer Invasion That Acts via the Modification of Metastasis-Associated Molecules

The actin-bundling protein, fascin, is a member of the cytoskeletal protein family that has restricted expression in specialized normal cells. However, many studies have reported the induction of this protein in various transformed cells including breast cancer cells. While the role of fascin in the regulation of breast cancer cell migration has been previously shown, the underlying molecular mechanism remained poorly defined. We have used variety of immunological and functional assays to study whether fascin regulates breast cancer metastasis-associated molecules. In this report we found a direct relationship between fascin expression in breast cancer patients and; metastasis and shorter disease-free survival. Most importantly, in vitro interference with fascin expression by loss or gain of function demonstrates a central role for this protein in regulating the cell morphology, migration and invasion potential. Our results show that fascin regulation of invasion is mediated via modulating several metastasis-associated genes. We show for the first time that fascin down-regulates the expression and nuclear translocation of a key metastasis suppressor protein known as breast cancer metastasis suppressor-1 (BRMS1). In addition, fascin up-regulates NF-kappa B activity, which is essential for metastasis. Importantly, fascin up-regulates other proteins that are known to be critical for the execution of metastasis such as urokinase-type plasminogen activator (uPA) and the matrix metalloproteases (MMP)-2 and MMP-9. This study demonstrates that fascin expression in breast cancer cells establishes a gene expression profile consistent with metastatic tumors and offers a potential therapeutic intervention in metastatic breast cancer treatment through fascin targeting

Differential marker expression by cultures rich in mesenchymal stem cells

Background: Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. Results: Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers – CD24, CD108 and CD40. Conclusion: We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers

FOXP3+ Tregs and B7-H1+/PD-1+ T lymphocytes co-infiltrate the tumor tissues of high-risk breast cancer patients: Implication for immunotherapy

<p>Abstract</p> <p>Background</p> <p>Recent studies have demonstrated a direct involvement of B7-H1, PD-1 and FOXP3 molecules in the immune escape of cancer. B7-H1 is an inhibitory molecule that binds to PD-1 on T lymphocytes, while FOXP3 is a marker for regulatory T cells (T_regs). We have previously demonstrated the association of B7-H1-expressing T infiltrating lymphocytes (TIL) with high-risk breast cancer patients while other studies reported the involvement of FOXP3+ T_regsas a bad prognostic factor in breast tumors. Although the co-existence between the two types of cells has been demonstrated <it>in vitro </it>and animal models, their relative infiltration and correlation with the clinicopathological parameters of cancer patients have not been well studied. Therefore, we investigated TIL-expressing the B7-H1, PD-1, and FOXP3 molecules, in the microenvironment of human breast tumors and their possible association with the progression of the disease.</p> <p>Methods</p> <p>Using immunohistochemistry, tumor sections from 62 breast cancer patients were co-stained for B7-H1, PD-1 and FOXP3 molecules and their expression was statistically correlated with factors known to be involved in the progression of the disease.</p> <p>Results</p> <p>A co-existence of B7-H1⁺T lymphocytes and FOXP3⁺T_regswas evidenced by the highly significant correlation of these molecules (<it>P </it>< .0001) and their expression by different T lymphocyte subsets was clearly demonstrated. Interestingly, concomitant presence of FOXP3⁺T_regs, B7-H1⁺and PD-1⁺TIL synergistically correlated with high histological grade (III) (<it>P </it>< .001), estrogen receptor negative status (<it>P </it>= .017), and the presence of severe lymphocytic infiltration (<it>P </it>= .022).</p> <p>Conclusion</p> <p>Accumulation of TIL-expressing such inhibitory molecules may deteriorate the immunity of high-risk breast cancer patients and this should encourage vigorous combinatorial immunotherapeutic approaches targeting T_regsand B7-H1/PD-1 molecules.</p

PD-L1 is overexpressed on breast cancer stem cells through notch3/mTOR axis

The T-cell inhibitory molecule PD-L1 is expressed on a fraction of breast cancer cells. The distribution of PD-L1 on the different subpopulations of breast cancer cells is not well-defined. Our aim was to study the expression level of PD-L1 on breast cancer stem-like (CSC-like) cells and their differentiated-like counterparts. We used multi-parametric flow cytometry to measure PD-L1 expression in different subpopulations of breast cancer cells. Pathway inhibitors, quantitative immunofluorescence, cell sorting, and western blot were used to investigate the underlying mechanism of PD-L1 upregulation in CSC-like cells. Specifically, PD-L1 was overexpressed up to three folds on breast CSC-like cells compared with more differentiated-like cancer cells. Functional in vitro and in vivo assays show higher stemness of PD-L1hi as compared with PD-L1lo cells. Among different pathways examined, PD-L1 expression on CSCs was partly dependant on Notch, and/or PI3K/AKT pathway activation. The effect of Notch inhibitors on PD-L1 overexpression in CSCs was completely abrogated upon mTOR knockdown. Specific knockdown of different Notch receptors shows Notch3 as a mediator for PD-L1 overexpression on CSCs and important for maintaining their stemness. Indeed, Notch3 was found to be overexpressed on PD-L1hi cells and specific knockdown of Notch3 abolished the effect of notch inhibitors and ligands on PD-L1 expression as well as mTOR activation. Our data demonstrated that overexpression of PD-L1 on CSCs is partly mediated by the notch pathway through Notch3/mTOR axis. We propose that these findings will help in a better design of anti-PD-L1 combination therapies to treat breast cancer effectively