Inhibition Of CD44 Expression Research Articles

Knockdown of ALDH1A3 reduces breast cancer stem cell marker CD44 via the miR-7-TGFBR2-Smad3-CD44 regulatory axis.

Inhibition of aldehyde dehydrogenase 1 family member A3 (ALDH1A3) has been revealed to lead to significant increase of microRNA (miR)-7 expression and decrease of CD44 expression in breast cancer stem cells (BCSCs), however the mechanism is not clear. The aim of the present study was to investigate the regulatory relationship between ALDH1A3, miR-7, and CD44 in BCSCs. The expression of ALDH1A3 was inhibited by small interfering RNA (siRNA or si), and the expression of miR-7 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Then, the ratio of CD44+ cells was analyzed by flow cytometry in MDA-MB-231 cells. The dual-luciferase reporter system was used to demonstrate that miR-7 binds to transforming growth factor-β receptor 2 (TGFBR2) 3'UTR, and ChIP-PCR determined whether the transcription factor Smad3 binds to the upstream regulatory region of the CD44 promoter. The results revealed that siALDH1A3 downregulated ALDH1A3 and promoted miR-7 expression, which resulted in downregulation of CD44 expression. siALDH1A3 also downregulated the CD44 expression on the surface of MDA-MB-231 cells and inhibited the G2/M phase in BCSCs as analyzed by flow cytometry. In addition, lenti-miR-7 cells transfected with TGF-β1 + SB431542 revealed that lenti-miR-7 inhibited the TGF-β1 pathway by inhibiting Smad2/3/4 expression and, thus, downregulated CD44 expression. miR-7 was revealed to directly bind to the TGFBR2 3'UTR through dual-luciferase reporter assay, and Smad3, a transcription factor, through ChIP-PCR was demonstrated to bind to the upstream region of the CD44 promoter. These results demonstrated the existence of the ALDH1A3-miR-7-TGFBR2-Smad3-CD44 axis in MDA-MB-231 cells. RT-qPCR results of 12 breast cancer surgical specimens and SK-BR-3, MCF-7, and LD cell lines further confirmed the presence of the regulatory axis. In conclusion the findings from the present study demonstrated that the ALDH1A3-miR-7-TGFBR2-Smad3-CD44 regulatory axis was highly efficient in the inhibition of CD44 expression in BCSCs, and that the regulatory expression of ALDH1A3 and miR-7 may provide a strategy in the therapy of breast cancer.

Effect of CD44 on differentiation of human amniotic mesenchymal stem cells into chondrocytes via Smad and ERK signaling pathways

CD44 antigen (CD44) is a transmembrane protein found in cell adhesion molecules and is involved in the regulation of various physiological processes in cells. It was hypothesized that CD44 directly affected the chondrogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs). In the present study, the expression of chondrocyte-associated factors was detected in the absence and presence of the antibody blocker anti-CD44 antibody during the chondrogenic differentiation of hAMSCs. Following inhibition of CD44 expression, the transcriptional levels of chondrocyte-associated genes SRY-box transcription factor 9, aggrecan and collagen type II α 1 chain, as well as the production of chondrocyte markers type II collagen and aggrecan were significantly decreased in hAMSCs. Further investigation indicated that there was no significant change in total ERK1/2 expression following inhibition of CD44 expression; however, phosphorylated (p)-ERK1/2 expression was decreased. The expression of p-Smad2/3 was also upregulated following CD44 inhibition. These data indicated that CD44 may affect the differentiation of hAMSCs into chondrocytes by regulating the Smad2/3 and ERK1/2 signaling pathway.

CD44 promotes cell proliferation in non-small cell lung cancer.

Cluster of differentiation 44 (CD44), a marker for cancer stem cells, has been reported to be associated with poor prognosis in non-small cell lung cancer (NSCLC), but its involvement in tumor growth has not fully been elucidated. The present study explored the associations between CD44 expression and clinicopathological features using immunohistochemistry in 94 NSCLC cases, as well as proliferation in cells with aberrant expression of this protein. Overexpression of CD44 was achieved by transfecting H1299 cells with CD44 expression vectors, and inhibition of CD44 expression was performed by transfecting small interfering RNAs into A549 cells. Cell proliferation was measured using Cell Counting Kit-8 assays and flat plate colony formation assay was performed to confirm the cellular anchorage growth in vitro. In total, 64 (68.1%) of the 94 NSCLC cases stained positive for CD44 expression. High expression of CD44 was associated with advanced T stage in NSCLC. Overexpression of CD44 in H1299 cells promoted cell proliferation and colony formation in vitro. Meanwhile, knockdown of CD44 expression in A549 cells suppressed cell proliferation and colony formation in vitro. High expression of CD44 may promote NSCLC progression by increasing cancer cell proliferation; therefore, it may serve as a potential biomarker for diagnosis or target therapy.

Combined SN-38 and gefitinib treatment promotes CD44 degradation in head and neck squamous cell carcinoma cells.

The aim of the present study was to search for an effective regimen among existing chemotherapies for head and neck squamous cell carcinoma (HNSCC). Among the tested drugs, we focused on combined SN-38, which is the active metabolite produced from irinotecan hydrochloride - a typeI DNA topoisomerase inhibitor - after it is metabolized by carboxylesterase in the liver and gefitinib, an EGFR tyrosine kinase inhibitor treatment, based on the ability of this combination to inhibit HNSCC cell growth. Contrary to our expectation, invivo, there was no significant difference in tumor growth suppression between gefitinib-only treatment and gefitinib plus SN-38. However, when tumor measurements were continued after treatment ceased, we found that several tumors showed renewed growth in the gefitinib-only group. The tumors that resumed growing after treatment showed increased CD44 expression compared with tumors from the combined treatment group. Next, we investigated the mechanism whereby SN-38 and gefitinib inhibited CD44 expression invitro. These studies revealed that the combined treatment promoted lysosomal degradation of CD44. The present study revealed that combined gefitinib and SN-38 treatment inhibits CD44 expression by promoting its lysosomal degradation in HNSCC cells. However, it is still unclear whether inhibition of CD44 expression in HNSCC cells can directly suppress tumor regrowth after therapy. Thus, it may be necessary to elucidate the relationship between the effects of these chemotherapeutic agents on CD44 expression and tumor recurrence/metastasis in future studies.

Perspectives of CD44 targeting therapies.

CD44 is a family of single-span transmembrane glycoproteins. Members of this family differ in the extracellular domain where ten variant exons are either excluded or included in various combinations. CD44 isoforms participate in many physiological processes including hematopoiesis, regeneration, lymphocyte homing and inflammation. Most importantly, they are involved in pathological processes and in particular in cancer. In several types of tumors, CD44 together with other antigens specifies for cancer stem cell populations. Mechanistically, CD44 proteins act as receptors for hyaluronan, co-receptor for receptor tyrosine kinases (RTKs) or G-protein-coupled receptors or provide a platform for metalloproteinases. For all these reasons, targeting CD44 may be a successful approach in cancer therapy. In this review, we discuss the various possibilities of targeting CD44. Among these are the production of CD44 ectodomains, antibodies, peptides or aptamers. Also inhibition of CD44 expression has been proposed. Finally, the function of CD44 as a hyaluronan receptor was also taken advantage of. We are convinced that the success of these therapies will depend on an increased understanding of the molecular functions of specific CD44 isoforms in particular in cancer stem cells.

Abstract 1969: Overexpression of specific CD44 variants mediate endocrine insensitivity and invasion in breast cancer cells

Abstract The majority of breast cancers express the oestrogen receptor and are potentially amenable to antihormone therapy; however the clinical effectiveness of these agents is limited by the phenomenon of acquired resistance which is associated with disease relapse and poor prognosis. In vitro evidence suggests that acquired resistance to tamoxifen and fulvestrant results in a significant gain to the migratory and invasive nature of breast cancer cells which may augment their metastatic potential in vivo. We have previously demonstrated that the CD44 receptor is overexpressed in acquired endocrine resistance and contributes to a gain in aggressive phenotype and poor outcome clinically. However, little is known regarding the effects of CD44 splice variants in this context. Here we have explored the role of CD44 variant 3 (v3) and CD44 variant 6 (v6) in endocrine-resistant and endocrine-sensitive breast cancer models. CD44v3 and CD44v6 isoform expression was assessed in cell models of acquired tamoxifen (Tam-R) and fulvestrant (Fas-R) resistance along with their endocrine-sensitive MCF-7 counterparts by microarray analysis, Western blotting, immunofluorescence and immunohistochemistry. The ability of CD44v3 and CD44v6 to modulate tumor cell growth (cell counting assay), migration (fibronectin motility assay), invasion (Matrigel Boyden chamber assay), adhesion (matrix adhesion assay) and response to endocrine agents were determined using cells in which CD44v3 or CD44v6 expression was modulated by overexpression (MCF-7) or by siRNA (Tam-R, Fas-R). Changes in cellular signalling pathways as a result of CD44 modulation and activation were assessed using Western blotting. CD44v3 and CD44v6 were overexpressed at gene and protein level in Tam-R and Fas-R cells versus endocrine-sensitive MCF-7 cells. Inhibition of CD44 expression (all isoforms) in resistant cells resulted in a loss of migratory, proliferative and invasive phenotype. Overexpression of CD44v6 in MCF-7 cells resulted in a gain in EGFR signalling and enhanced their intrinsic invasive capacity and promoted a loss of endocrine response. In contrast, overexpression of CD44v3 decreased EGFR pathway activity in MCF-7 cells and did not augment their invasive capacity or alter their endocrine sensitivity. Our data suggests that upregulation of CD44v6 in acquired resistant breast cancer may contribute to their gain in aggressive phenotype and loss of endocrine response through transactivation of the EGFR pathway. Future therapeutic targetting of CD44v6 may prove to be an effective strategy alongside EGFR targeted agents in delaying/preventing acquired resistance in breast cancer. Citation Format: Rebecca L. Bellerby, Christopher Smith, Julia Gee, Tracey Martin, Peter Barrett-Lee, Stephen Hiscox. Overexpression of specific CD44 variants mediate endocrine insensitivity and invasion in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1969. doi:10.1158/1538-7445.AM2014-1969

Overexpression of CD44 in acquired endocrine resistant breast cancer modulates erbB activity and promotes an invasive phenotype.

Abstract Abstract #3027 Background: Acquired endocrine resistance in breast cancer cells is accompanied by altered growth factor receptor signalling and a highly migratory cell phenotype. In tamoxifen-resistant ('TamR') MCF7 cells, our microarray analysis has demonstrated elevated levels of CD44, a transmembrane glycoprotein, known to interact with and modulate the function of a number of growth factor receptors and plays a major role in tumour metastasis. Here, we have explored the role of CD44 as a modulator of erbB family activity in TamR cells and evaluated the expression of CD44 in a series of breast cancer patients on endocrine therapy.
 Methods: RT-PCR was performed to confirm overexpression of CD44 at the gene level in TamR cells. Western blotting and immunocytochemistry were used to investigate expression of CD44 at the protein level together with the activity of erbB signalling pathways following stimulation of CD44 by its ligand, hyaluronan (HA), or following stimulation with erbB ligands (TGFα, heregulin β1) after knockdown of CD44 expression using siRNA. Immunofluorescence microscopy was used to determine association between CD44 and erbB family members. Cell migration was assessed by seeding cells onto fibronectin-coated microporous inserts for 24 hours. CD44 expression levels were evaluated using paraffin-embedded tissue sections in a series of primary breast cancer tissues (n=103) from patients receiving endocrine therapy by immunohistochemical staining.
 Results: CD44 was overexpressed at the gene and protein level in TamR cells versus their endocrine sensitive counterparts. siRNA-mediated suppression of CD44 expression reduced basal EGFR/Her2 activity in TamR cells together with suppressing their migratory phenotype. siRNA-mediated inhibition of CD44 expression greatly attenuated the ability of erbB ligands to activate erbB receptors and inhibited TGFα and heregulin β1-induced migration. Conversely, stimulation of TamR, but not MCF7 cells, with HA promoted activation of erbB family members together with their downstream signalling intermediates ERK1/2, Src and AKT. Analysis of CD44 expression in clinical tissue revealed an association between elevated CD44 expression and a shortened response to endocrine therapy.
 Conclusions: These data demonstrate that overexpression of CD44 accompanies endocrine resistance where it enhances the sensitivity of these cells to factors (erbB ligands, HA) found within the tumour microenvironment. This may implicate CD44 in loss of endocrine response clinically and, therefore, provide a novel therapeutic target in endocrine resistant breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3027.

Inactivation of the NF2 tumor suppressor protein merlin in DU145 prostate cancer cells

The neurofibromatosis 2 (NF2) tumor suppressor gene product merlin is an important regulator of contact-dependent cell proliferation. Phosphorylation of merlin at serine 518 (Ser518) by the Rac effector p21-activated kinase (PAK) inactivates merlin's growth suppressing function, and is regulated by cell-culture conditions, including cell density, cell/substrate attachment, and growth factor availability. We examined the regulation of merlin expression and merlin phosphorylation in prostate cancer cells. Phosphorylation of merlin in five prostate cancer cell lines (LNCaP, DU145, PC3, 22RV1, and LAPC-4) was examined by Western blotting using anti-phospho-merlin (Ser518) antibody. The activity of PAK, an upstream regulator of merlin phosphorylation, was measured by Western blotting using phospho-PAK (Ser141) antibody. The effects of various cell-culture conditions on the phosphorylation levels of merlin and PAK were analyzed. Both merlin expression and phosphorylation were low in LNCaP, PC3, 22RV1, and LAPC-4 prostate cancer cells. In DU145 cells, total and phosphorylated merlin were abundant, but phosphorylation was not inhibited by high cell density, serum withdrawal, the addition of hyaluronic acid or inhibition of CD44 expression, all of which are reported to inhibit merlin phosphorylation in non-neoplastic cells. PAK activation was elevated in DU145 cells and the addition of a PAK-specific inhibitor peptide but not the Rac1-specific inhibitor NSC23766 inhibited both PAK and merlin phosphorylation. Merlin is inactivated in DU145 prostate cancer cells by PAK-mediated constitutive phosphorylation, identifying a novel mechanism of merlin inactivation in neoplastic cells.

Inhibition of CD44 expression in hepatocellular carcinoma cells enhances apoptosis, chemosensitivity, and reduces tumorigenesis and invasion

CD44 is overexpressed in various tumors including hepatocellular carcinoma (HCC). The purpose of this study was to examine the effects of CD44 antisense oligonucleotide (ASO) alone or combination with doxorubicin on HCC cells in vitro. Cytotoxicity was measured by use of a cell viability assay in HCC cell line SNU-449. Tumorigenesis and invasion were accessed by colony formation, growth in soft agar and ECMatrix invasion assay. Apoptosis and necrosis were evaluated by using double staining with Hoechst 33342 and propidium iodide. Protein expression and mRNA level were detected by Western blot and RT-PCR. We have designed novel CD44 ASO, which can effectively down-regulate CD44 expression in SNU-449. Colony formation, growth in soft agar and invasion were significantly impaired after CD44 ASO treatment in SNU-499. In company with CD44 down-regulated by CD44 ASO, MDR-1 and Bcl-2 expression were also greatly reduced. CD44 ASO also increased chemosensitivity to doxorubicin significantly, lowered IC(50 )by one order of magnitude. Apoptosis and necrosis were also induced by CD44 ASO alone or in combination treatment with doxorubicin. Inhibition of CD44 expression by CD44 ASO significantly induced apoptosis, decreased tumorigenesis and invasion, and increased chemosensitivity. Thus, CD44 ASO is potentially a therapy that is worth investigating in the clinical setting.

Suppression of human colon cancer tumors in nude mice by siRNA CD44 gene therapy

The expression of CD44, an adhesion protein associated with the tumor stem cell phenotype, is increased in most human malignant neoplasms. To further delineate the role of CD44 in colon cancer, we inhibited its expression using the siRNA method. HT-29, a human colon cancer cell line producing a large amount of CD44, was transfected with a construct producing a siRNA targeting a 19 mer sequence of the transmembrane domain of CD44 spanning between exon 16 and 17. Following stable transfection, siRNA CD44 resulted in over 75% inhibition of CD44 expression. The stable lines were less adhesive to hyaluronan and more susceptible to apoptosis induced by etoposide. siRNA CD44 clones formed a lower number and size of colonies in soft agar assays. A siRNA CD44 cell clone xenografted in nude mice generated tumors with a reduced tumor volume and wet weight, as compared to control vector clone. Intratumoral gene therapy with a polyethylenimine/siRNA CD44 plasmid DNA complex resulted in tumor growth suppression in nude mice. After siRNA CD44 intratumoral gene therapy, apoptosis was increased in the tumors when compared to the control vector group. In conclusion, based on this mouse xenograft model, siRNA targeting a discrete sequence of human CD44 may provide a potential therapeutic option for colon cancer.

Bromelain protease F9 reduces the CD44 mediated adhesion of human peripheral blood lymphocytes to human umbilical vein endothelial cells

The thiol protease bromelain has been shown to remove T-cell CD44 molecules from lymphocytes and to affect T-cell activation. We investigated the effect of a highly purified bromelain protease F9 (F9) on the adhesion of peripheral blood lymphocytes (PBL) to human umbilical vein endothelial cells (HUVEC). Preincubation of the lymphocytes with F9 reduced the adherence to about 20% of unstimulated and to about 30% of phorbol-dibutyrate (P(Bu)2) stimulated lymphocytes. Using flow cytometry, both crude bromelain and protease F9 reduced the expression of CD44, but not of LFA-1, on PBL. F9 was about 10 times more active than crude bromelain; at 2.5 micrograms/ml of F9 about 97% inhibition of CD44 expression was found. A mAb against CD44 was tested and found to block the F9-induced decrease in PBL-binding to HUVEC. The results indicate that F9 selectively decreases the CD44 mediated binding of PBL to HUVEC.

Detection, localization and modulation of hyaluronic acid/CD44 receptor expression in patients with endocrine orbitopathy

Accumulation of hyaluronic acid within the orbital tissues represents a histological hallmark of Graves' ophthalmopathy. The hyaluronic acid/CD44-receptor plays a key role in the binding and metabolism of hyaluronic acid, and affects numerous cellular functions of potential relevance to the pathogenesis of Graves' ophthalmopathy, including cell proliferation, migration, and adhesive interactions between connective tissue components and immunocompetent cells. Using a highly sensitive immunoperoxidase technique and monoclonal antibodies directed against the standard CD44 molecule, we examined the expression and modulation of hyaluronic acid/CD44 receptors in cryostat sections of orbital biopsy specimens derived from patients with severe Graves' ophthalmopathy and normal individuals. Modulation of CD44 by cytokines and affinity-purified IgGs derived from patients with Graves' ophthalmopathy was studied in extracts of fibroblast monolayers following stimulation in vitro, using SDS-polyacrylamid gel electrophoresis and immunoblotting. Strong immunoreactivity for CD44 was present in all specimens derived from patients with Graves' ophthalmopathy and was detected in fibroblasts residing in retroorbital connective tissue, in extracellular matrix components and in mononuclear cell infiltrates. By contrast, in normal orbital specimens, CD44 immunoreactivity was faint and present only in occasional connective tissue cells. Recombinant IL-1 alpha, TNF alpha, IGF-1 and GO-IgGs significantly stimulated CD44 expression in Graves' retroocular fibroblasts (range: 168 to 588%; all p < 0.05). By contrast, EGF, IL-6, control IgGs and 15% fetal calf serum failed to alter CD44 expression. Treatment of monolayers with IFN gamma resulted in weak inhibition of CD44 expression. In conclusion, the hyaluronic acid/CD44 receptor is expressed at elevated levels in Graves' orbital connective tissue in situ.(ABSTRACT TRUNCATED AT 250 WORDS)