Decreased CD44v3 Expression Impairs Endometrial Stromal Cell Decidualization in Women With Recurrent Implantation Failure

BackgroundThe precise pathogenesis of poor endometrial receptivity in recurrent implantation failure (RIF) still remains unclear. This study aims to explore the effects of different CD44 isoforms in the mid-secretory phase endometrium on endometrial receptivity in women with RIF.MethodsMid-secretory phase endometrial tissue samples were obtained from two groups of women who had undergone IVF: a) 24 patients with RIF, b) 18 patients with infertility due to tubal obstruction, who had achieved a successful clinical pregnancy after the first embryo transfer in IVF (control group). Identification of differentially expressed CD44 isoforms in endometrial tissues was assessed with immunohistochemistry, qPCR and western blotting. Effects of CD44v3 overexpression and knockdown on proliferation and decidualization of Immortalized human endometrial stromal cells (T-HESCs) and primary HESCs were investigated by qPCR and Western blot. A heterologous co-culture system of embryo implantation was constructed to mimics the process of trophoblast invasion during implantation.ResultsCD44v3 was significantly higher expressed in mid-secretory phase of endometrial stromal cells than proliferation phase, but was notably lower in RIF patients. The expression of decidualization markers, prolactin (PRL) and insulin like growth factor binding protein-1 (IGFBP1), was notably decreased following CD44v3 knockdown, whereas the expression levels of both PRL and IGFBP1 increased after CD44v3 overexpression in HESCs. Furthermore, the CD44v3-knockdown HESCs displayed a significantly deficiency in supporting trophoblast outgrowth through a co-culture system of embryo implantation; however, CD44v3 overexpression in HESCs promoted trophoblast outgrowth.ConclusionThe reduced expression of CD44v3 suppresses HESCs proliferation and decidualization, which might play a pivotal role in poor endometrial receptivity in women with RIF.

New monoclonal antibodies that recognize an unglycosylated, conserved, extracellular region of CD44 in vitro and in vivo, and can block tumorigenesis

CD44 is a transmembrane glycoprotein that binds to hyaluronic acid, plays roles in a number of cellular processes and is expressed in a variety of cell types. It is up-regulated in stem cells and cancer. Anti-CD44 monoclonal antibodies affect cell motility and aggregation, and repress tumorigenesis and metastasis. Here we describe four new anti-CD44 monoclonal antibodies originating from B cells of a mouse injected with a plasmid expressing CD44 isoform 12. The four monoclonal antibodies bind to the terminal, extracellular, conserved domain of CD44 isoforms. Based on differences in western blot patterns of cancer cell lysates, the four anti-CD44 mAbs separated into three distinct categories that include P4G9, P3D2, and P3A7, and P3G4. Spot assay analysis with peptides generated inEscherichia colisupport the conclusion that the monoclonal antibodies recognize unglycosylated sequences in the N-terminal conserved region between amino acid 21–220, and analyses with a peptide generated in human embryonic kidney 293 cells, demonstrate that these monoclonal antibodies bind to these peptides only after deglycosylation. Western blots with lysates from three cancer cell lines demonstrate that several CD44 isoforms are unglycosylated in the anti-CD44 target regions. The potential utility of the monoclonal antibodies in blocking tumorigenesis was tested by co-injection of cells of the breast cancer-derived tumorigenic cell line MDA-MB-231 with the anti-CD44 monoclonal antibody P3D2 into the mammary fat pads of mice. All five control mice injected with MDA-MB-231 cells plus anti-IgG formed palpable tumors, while only one of the six test mice injected with MDA-MB-231 cells plus P3D2 formed a tiny tumor, while the remaining five were tumor-free, indicating that the four anti-CD44 mAbs may be useful therapeutically.

In Silico Design and Selection of New Tetrahydroisoquinoline-Based CD44 Antagonist Candidates

CD44 promotes metastasis, chemoresistance, and stemness in different types of cancer and is a target for the development of new anti-cancer therapies. All CD44 isoforms share a common N-terminal domain that binds to hyaluronic acid (HA). Herein, we used a computational approach to design new potential CD44 antagonists and evaluate their target-binding ability. By analyzing 30 crystal structures of the HA-binding domain (CD44HAbd), we characterized a subdomain that binds to 1,2,3,4-tetrahydroisoquinoline (THQ)-containing compounds and is adjacent to residues essential for HA interaction. By computational combinatorial chemistry (CCC), we designed 168,190 molecules and compared their conformers to a pharmacophore containing the key features of the crystallographic THQ binding mode. Approximately 0.01% of the compounds matched the pharmacophore and were analyzed by computational docking and molecular dynamics (MD). We identified two compounds, Can125 and Can159, that bound to human CD44HAbd (hCD44HAbd) in explicit-solvent MD simulations and therefore may elicit CD44 blockage. These compounds can be easily synthesized by multicomponent reactions for activity testing and their binding mode, reported here, could be helpful in the design of more potent CD44 antagonists.

Expression of CD44 Isoforms in Human Colorectal Tumor Cell Lines

The expression profile of CD44 isoforms in 55 colorectal cancer (CRC) cell lines has been evaluated based on mRNA sequencing data from the Cancer Cell Line Encyclopedia database. The distribution of CD44 mRNA isoforms differs significantly between CRC lines. In 13 studied lines, including Caco-2 and RKO, CD44 expression was not detected. In most other lines CD44 mRNA isoform 3 was the most abundant; however, the level of its expression varied, and it was absent in the MDST8 and SNU503 lines. The highest level of isoform 3 was observed in CW2 and T84 lines. The next most frequent was isoform 4 with its expression level lower than that of isoform 3, except for the HCT116, SNU81, NCIH508 and SNUC4 lines. The highest expression of isoform 4 was detected in the MDST8 line, the only line in which isoform 6 was also expressed. Isoform 2 was also present in CRC cell lines; its highest expression level was found in the SNU503 line. Isoforms 1, 5, and 7 did not show expression in any of the studied lines. It is necessary to take into account the mRNA expression profile of specific CD44 isoforms when choosing a cell model to study its role in CRC. colorectal cancer, CD44 isoforms, mRNA sequencing, CCLE The study was funded by the Russian Science Foundation (project no. 17-14-01338).

A Fluorescent Aptasensor based on Copper Nanoclusters for Optical Detection of CD44 Exon v10; an important isoform in metastatic breast cancer

Recent studies suggest that breast cancer cells express various CD44 isoforms. CD44 is an integral transmembrane protein encoded by a single 20-exon gene. Exon v10 of CD44 plays a key...

Skipping Exon-v6 from CD44v6-Containing Isoforms Influences Chemotherapy Response and Self-Renewal Capacity of Gastric Cancer Cells

De novo expressed CD44 isoforms containing exon-v6 are frequently associated with gastric cancer (GC) aggressiveness, and may predict chemotherapy response in vitro. Whether exon-v6 itself is responsible for conferring these properties to CD44v6-containing isoforms remains to be elucidated. CRISPR/Cas9 and Phosphorodiamidate Morpholino oligomers (PMOs) were used to induce specific exon-v6 skipping, maintaining the CD44 reading frame, in two GC cell lines endogenously expressing CD44v6. Cisplatin and 5-fluorouracil treatment response, and self-renewal ability was compared between CRISPR/Cas9-edited, CD44v6 knockdown and mock cells. We obtained homozygous genome-edited cell lines with exon-v6 deletion. Edited cells transcribed CD44v isoforms presenting in frame v5–v7 splicing, mimicking exon-v6 skipping. Results showed that removing specifically exon-v6 sensitizes cells to cisplatin and impairs cells’ self-renewal ability, similarly to CD44v6 knockdown. In parallel, we also tested a clinically feasible approach for transient exon-v6 skipping with a PMO-based strategy. We demonstrate that exon-v6 specific removal from CD44v isoforms increases cell sensitivity to cisplatin and impairs GC cells self-renewal. We trust that a PMO approach designed towards CD44v6 overexpressing GC cells may be a suitable approach to sensitize tumor cells for conventional therapy.

Impact of Truncated O-glycans in Gastric-Cancer-Associated CD44v9 Detection

CD44 variant isoforms are often upregulated in cancer and associated with increased aggressive tumor phenotypes. The CD44v9 is one of the major protein splice variant isoforms expressed in human gastrointestinal cancer cells. Immunodetection of CD44 isoforms like CD44v9 in tumor tissue is almost exclusively performed by using specific monoclonal antibodies. However, the structural variability conferred by both the alternative splicing and CD44 protein glycosylation is disregarded. In the present work, we have evaluated the role of O-glycosylation using glycoengineered gastric cancer models in the detection of CD44v9 by monoclonal antibodies. We demonstrated, using different technical approaches, that the presence of immature O-glycan structures, such as Tn and STn, enhance CD44v9 protein detection. These findings can have significant implications in clinical applications mainly at the detection and targeting of this cancer-related CD44v9 isoform and highlight the utmost importance of considering glycan structures in cancer biomarker detection and in therapy targeting.

Epithelial-To-Mesenchymal Transition Markers and CD44 Isoforms Are Differently Expressed in 2D and 3D Cell Cultures of Prostate Cancer Cells

Three-dimensional (3D) cell cultures allow the mimic of functions of living tissues andprovide key information encoded in tissue architecture. Considered the pivotal role of epithelial-tomesenchymaltransition (EMT) in carcinoma progression, including prostate cancer (PCa), weaimed at investigating the effect of the 3D arrangement on the expression of some key markers ofEMT in cultured human prostate cancer (PCa) cells, to better understand PCa cell behavior. PC3 andDU145 PCa cells were cultured in RPMI cell culture medium either in 2D-monolayers or in 3Dspheroids.The main EMT markers E-cadherin, N-cadherin, α-smooth muscle actin (αSMA),vimentin, Snail, Slug, Twist and Zeb1 were evaluated by confocal microscopy, real-time PCR andWestern blot. Confocal microscopy revealed that E-cadherin was similarly expressed at the cellboundaries on the plasma membrane of PCa cells grown in 2D-monolayers, as well as in 3Dspheroids,but resulted up-regulated in 3D-spheroids, compared to 2D-monolayers, at the mRNAand protein level. Moreover, markers of the mesenchymal phenotype were expressed at very lowlevels in 3D-spheroids, suggesting important differences in the phenotype of PCa cells grown in 3Dspheroidsor in 2D-monolayers. Considered as a whole, our findings contribute to a clarification ofthe role of EMT in PCa and confirm that a 3D cell culture model could provide deeper insight intothe understanding of the biology of PCa.

Overexpression of CD44 Variant 9: A Novel Cancer Stem Cell Marker in Human Cholangiocarcinoma in Relation to Inflammation

Various CD44 isoforms are expressed in several cancer stem cells during tumor progression and metastasis. In particular, CD44 variant 9 (CD44v9) is highly expressed in chronic inflammation-induced cancer. We investigated the expression of CD44v9 and assessed whether CD44v9 is a selective biomarker of human cholangiocarcinoma (CCA). The expression profile of CD44v9 was evaluated in human liver flukeOpisthorchis viverrini-related CCA (OV-CCA) tissues, human CCA (independent of OV infection, non-OV-CCA) tissues, and normal liver tissues. CD44v9 overexpression was detected by immunohistochemistry (IHC) in CCA tissues. There was a higher level of CD44v9 expression and IHC score in OV-CCA tissues than in non-OV-CCA tissues, and there was no CD44v9 staining in the bile duct cells of normal liver tissues. In addition, we observed significantly higher expression of inflammation-related markers, such as S100P and COX-2, in OV-CCA tissues compared to that in non-OV and normal liver tissues. Thus, these findings suggest that CD44v9 may be a novel candidate CCA stem cell marker and may be related to inflammation-associated cancer development.