Abstract. CD44, a major receptor for hyaluronan (HA), is a member of a class of adhesion molecules of unknown classification involved in cell proliferation, differentiation, migration, angiogenesis, and the presentation of specific cytokines to the corresponding receptors as well as in cell signaling transduction. It has recently been discovered that CD44, a marker of tumor stem cells, is involved in the drug resistance and invasion of multiple types of tumors. The 20 exons in the CD44 gene that are alternatively spliced, give rise to many CD44 isoforms, possibly including tumor-specific sequences. Dozens of CD44 isoforms have been found, to date, and the standard CD44 (CD44s) isoform is the most common. We recently showed that a novel short-tail isoform of CD44 (CD44st) was expressed in multidrug-resistant human breast cancer MCF-7/Adr cells. Moreover, the novel CD44st was able to interact with HA and regulate the expression of matrix metalloproteinase (MMP)-2 and MMP-9, which increased the invasive capability of MCF-7 cells through the Ras/MAPK signaling pathway. In the present study, we verified that MCF-7 cells subjected to drug pressure develop multidrug resistance to doxorubicin, and the expression levels of multidrug resistance protein 1 (MDR1), CD44st and nuclear factor-κB (NF-κB) mRNA and protein were gradually upregulated in a dose-dependent manner in MCF-7 cells treated with doxorubicin. HA increases the secretion of MMP-2 and MMP-9 in multidrug-resistant MCF-7 cells and affected the invasive ability of MCF-7 cells through the upregulation of CD44st expression, and such an effect was blocked by the NF-κB-specific inhibitor BMS-345541.

BackgroundCD44, a hyaluronan (HA) receptor, is a multistructural and multifunctional cell surface molecule involved in cell proliferation, cell differentiation, cell migration, angiogenesis, presentation of cytokines, chemokines and growth factors to the corresponding receptors, and docking of proteases at the cell membrane, as well as in signaling for cell survival. The CD44 gene contains 20 exons that are alternatively spliced, giving rise to many CD44 isoforms, perhaps including tumor-specific sequences.MethodsReverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to detect CD44st mRNA and CD44 protein in sensitive MCF-7, Lovo, K562 and HL-60 cell lines as well as their parental counterparts, respectively. The full length cDNA encoding CD44st was obtained from the total RNA isolated from MCF-7/Adr cells by RT-PCR, and subcloned into the pMD19-T vector. The CD44st gene sequence and open reading frame were confirmed by restriction enzyme analysis and nucleotide sequencing, and then inserted into the eukaryotic expression vector pcDNA3.1. The pcDNA3.1-CD44st was transfected into MCF-7 cells using Lipofectamine. After transfection, the positive clones were obtained by G418 screening. The changes of the MMP-2 and MMP-9 genes and protein levels were detected by RT-PCR and gelatin zymography, respectively. The number of the cells penetrating through the artificial matrix membrane in each group (MCF-7, MCF-7+HA, MCF-7/neo, MCF-7/neo+HA, MCF-7/CD44st, MCF-7/CD44st+HA and MCF-7/CD44st+Anti-CD44+HA) was counted to compare the change of the invasion capability regulated by the CD44st. Erk and P-Erk were investigated by Western blotting to approach the molecular mechanisms of MMP-2 and MMP-9 expression regulated by the CD44st.ResultsSensitive MCF-7, Lovo, K562 and HL-60 cells did not contain CD44st mRNA and CD44 protein. In contrast, the multidrug resistance MCF-7/Adr, Lovo/Adr, K562/Adr and HL-60/Adr cells expressed CD44st mRNA and CD44 protein. The CD44st mRNA gene sequence was successfully cloned into the recombinant vector pcDNA3.1 and identified by the two restriction enzymes. It was confirmed that the reconstructed plasmid contained the gene sequence of CD44st that was composed of exons 1 to 4, 16 to 17, and 1 to 205 bases of exons 18. The new gene sequence was sent to NCBI for publication, and obtained the registration number FJ216964. The up-regulated level of the mRNA of the CD44 gene and the CD44 protein were detected, respectively, by RT-PCR and flow cytometry in MCF-7 cells transfected with pcDNA3.1-CD44st. The invasiveness of the cells and the activity of MMP-2 and MMP-9 were clearly activated by HA treatment, and blocked by CD44 neutralizing antibody. MCF-7/CD44st cells pretreated with the neutralizing antibody against CD44, and the inhibitor of MAPKs signaling pathway, could strongly block the expression of P-Erk.ConclusionsA new CD44st was expressed in multidrug resistant MCF-7/Adr, Lovo/Adr, K562/Adr and HL-60/Adr cells. The expression vector pcDNA3.1-CD44st was cloned and ...

Background: Abnormal expression of some CD44 molecules in tumor tissues can induce the degradation of the extracellular matrix, and is closely associated with axillary lymph node metastasis, angiogenesis, cancer progression, and drug resistance. Patients and Methods: We measured and confirmed the expression of CD44st and MMP2 mRNAs and proteins in the cancer tissues and adjacent normal tissues of postoperative non-small cell lung cancer (NSCLC) patients with either adenocarcinoma (n = 72) or squamous cell carcinoma (n = 53) using quantitative reverse transcription polymerase chain reaction, gene sequencing, and immunohistochemical analysis. Results: CD44st and MMP2 expression were closely associated with the histopathological classification, lymph node metastasis, and TNM stage of the tumors, and the difference was statistically significant (p < 0.05). The median overall survival (OS) for the high CD44st expression group was 30.52 months (95% confidence interval (CI) 24.02-36.15); for the low expression group it was 43.23 months (95% CI 31.81-52.02) (p = 0.020). The median OS for the high MMP-2 expression group was 30.53 months (95% CI 26.69-33.31); for the low expression group it was 40.06 months (95% CI 33.55-46.45) (p = 0.022). Conclusion: The rates of CD44st and MMP2 expression were higher in squamous cell carcinomas than in adenocarcinomas, were closely associated with lymph node metastasis and TNM stage, and affected patients' prognoses.

ObjectiveCD44st is a member of the CD44 family; abnormal expression of some CD44 isoforms are closely associated with axillary lymph node metastasis, cancer progression, and patients’ prognosis. The objective of this study is to investigate the correlation between the expression of CD44st and HER2 in breast cancer and the effect on patients’ prognosis.MethodsPrimers were designed to target the CD44st mRNA (Gene Bank No FJ216964) which has been newly identified in a drug-resistant breast cancer cell line. The expression of CD44st and HER2 mRNA and proteins in cancerous and paracancerous tissue of postoperative breast cancer patients was detected and compared. Tissue samples were obtained from 102 cases of invasive ductal carcinoma, 19 cases of intraductal carcinoma, and 11 cases of medullary carcinoma. The correlation between CD44st and HER2 expression and clinical pathological features was examined.ResultsThe expression rate of CD44st mRNA and protein in breast cancer tissue was 64.4% (85/132), while HER2 mRNA and protein was expressed in 22.0% (29/106) of the samples. The expression of CD44st and HER2 were low in paracancerous tissue. In breast cancer tissue, the expression rate of HER2 mRNA and protein in the CD44st-positive group was 28.2% (24/85), and 10.6% (5/47) in the CD44st-negative group. This difference was statistically significant (P=0.015). Sequencing analysis showed that the amplified CD44st gene in this study was the same as that which was previously discovered in the drug-resistant breast cancer cell line. A linear correlation was found between the expression of CD44st and HER2 (r=0.972, r2=0.945, F=2,213.51, P<0.001). The expression of CD44st and HER2 was also closely associated with luminal cancer subtypes, lymph node metastasis, and TNM stage (P<0.05), but not associated with age, pathological type, or tumor size (P>0.05). The median overall survival in the CD44st high-expression group was 51.85 months (95% CI: 48.48–55.22), which was significantly shorter than that in the CD44st low-expression group (57.61 months; 95% CI: 55.54–59.68, P=0.032).ConclusionCD44st is closely related to the expression of HER2. The expression of CD44st affects patient prognosis and is associated with lymph node metastasis, TNM staging, and molecular subtyping.