cd44

Recent studies have indicated that assessing the expression levels of cancer stem cell markers is critical in predicting the behavior of these neoplasms. This study aimed to evaluate and compare the expression levels of CD44 and epithelial cell adhesion molecule (EpCAM) markers in pleomorphic adenoma (PA) and mucoepidermoid carcinoma (MEC) using immunohistochemistry.

In this cross‑sectional descriptive‑analytical study, 20 samples each of PA and MEC were selected from Kashani Hospital, Isfahan, Iran, based on inclusion and exclusion criteria. Specimens were prepared using immunohistochemical methods and analyzed under an optical microscope. Pathologists evaluated microscopic grade, staining intensity and percentage, and the staining intensity distribution (SID) index. Statistical analysis was conducted with SPSS (version 26), employing the Kolmogorov–Smirnov test, t‑test, Chi‑square, and Fisher’s exact test.

The mean frequency of stained cells for both CD44 (P = 0.39) and EpCAM (P = 0.40) markers showed no statistically significant differences between the PA and MEC groups. Similarly, the mean intensity of staining did not differ significantly for either CD44 (P = 0.40) or EpCAM (P = 0.18). The average SID index for the EpCAM marker in the MEC group was significantly higher than the PA group (P = 0.03) and for the EpCAM marker, there was a significant difference between the average SID index and all three variables of microscopic grade (P = 0.01), clinical stage (P = 0.00), and 3‑year prognosis (P = 0.02).

The use of EpCAM immunohistochemical marker may help to predict the behavior of salivary gland tumors and obtain better treatment measures for patients. 

The aim of the study is to compare the expression of CD44 in OLP, OL and OSCC.

Methods and Materials: 

This laboratory study is performed on paraffin blocks from tissue biopsies of patients with OLP, OL and OSCC available in the maxillofacial archive of pathology department of Imam Khomeini hospital, Urmia; 12 blocks of each lesion and 10 normal mucosal tissue blocks as a control group. The smears were stained by H&E and then stained by CD44 antibody kit and cut into 5-micron dimensions. 4 codes were used to evaluate the intensity of staining based on the average percentage of stained cells to total cells: staining less than five percent with a negative code, staining five to 25% code +1, staining 25% to 50 % code +2, staining 50% to 75% code +3 and for staining 75% and above code +4. Then the data was analyzed.

There is a significant difference between the three lesions of OLP, OL, and OSCC in the expression level of CD44 (p<0.001) and expression of this molecule in the mucosa of OSCC was higher compared to the OLP and OL. Also, the expression of CD44 in three lesions of OLP, OL, and OSCC had a statistically significant difference with oral normal mucosa (p<0.001).

Based on the findings, this protein can probably be a suitable marker to confirm the prediction of dysplasia in oral premalignant lesions such as OLP and OL, and invasion, metastasis, prognosis and recurrence in OSCC.

The study aims to evaluate CD44 expression as a cancer stem cell marker in Triple-negative breast cancer (TNBC) and its correlation with prognostic parameters. Evaluation of CD44 immunoexpression in TNBC is vital for understanding tumor aggressiveness and determining its prognostic value.

In thishospital-based cross-sectional study of 50 cases of primary triple negative breast cancer patients,the tissue sections were subjected to immunohistochemical examination using CD44 antigen marker. The proportion and intensity of CD44 immunostaining wereassessed and correlation with prognostic markers such as histological grade, tumor size and nodal statuswas examined.

CD44 expression was observed in 40% of the total cases with a statistically significant association with histological grade (P= 0.002). Higher CD44 expression was noticed with increasing tumour grade. However, no statistical significance was observed with respect to tumor size and nodal status.

The study suggests that CD44 immunoexpression may serve as a surrogate marker of BCSCs and may hold prognostic value in TNBC patients. However, further studies on larger samples are required to fully understand its role.

Several long non-coding RNAs are implicated in the development and metastasis of cancer. The non-coding RNA HOTAIR is known to play a significant role in the progression of various cancers.

This study aimed to assess the impact of HOTAIR dysregulation on stemness markers in AGS and MKN45 gastric cancer cell lines.

Downregulation and upregulation of HOTAIR were induced using small interfering RNA (siRNA) and a HOTAIR overexpression vector, respectively. The surface stemness markers CD24 and CD44 were analyzed using flow cytometry. Additionally, the effects on the expression of two stemness markers, NANOG and P21, were investigated using Quantitative Reverse Transcription PCR (qRT-PCR).

Flow cytometry analysis showed that HOTAIR modulates the cell-surface expression of CD44 and CD24 in gastric cancer cells. Furthermore, dysregulation of HOTAIR significantly affected the expression of the genes P21 and NANOG.

Our studies suggest that HOTAIR may regulate the stemness characteristics of gastric cancer cell lines.

CD44, a cell-surface receptor and a key player in cellular signaling, can act as both tumor suppressor and promoter. This study aimed to investigate the association of CD44 rs13347C>T variants with prostate neoplasms, including both benign prostatic hyperplasia (BPH) and prostate cancers using a case-control and bioinformatics approach. Genomic DNA was extracted from 545 blood samples (225 BPH, 225 prostate cancers, and 95 control) and the CD44 rs13347C>T genotypes were identified using PCR-RFLP. We explored miRNA interactions using the miRNASNP-v3 database and GeneMANIA for co-expression networks. Results showed cancer patients had significantly higher PSA levels compared to both controls (p= 0.03) and BPH (p= 0.01). Additionally, digital rectal examination-positive and smoker BPH patients showed significantly the increased cancer risk (p= 0.004, p= 0.046). Prostate cancer group indicated significantly higher frequency of CD44 rs13347C>T mutant allele compared to control and BPH groups, particularly in TT and CT+TT genotypes (p < 0.05). miRNA SNP-v3 database predicted the mutant allele of CD44 rs13347C>T could lose 1 and gain 6 miRNAs for a new site created. Co-expression analysis revealed a direct interaction between CD44 and aryl hydrocarbon receptor (AHR), a gene known to be dysregulated in smokers. Furthermore, these genes alone display co-expression interactions with integrin subunit alpha 4 (ITGA4), protein plays a paradoxical role, both suppressing and promoting tumors. Based on the findings, the mutant allele of CD44 rs13347C>T may disrupt miRNA binding, which may potentially impact CD44, AHR, and ITGA4 expression in smokers, possibly contributing to prostate cancer progression.

Breast cancer stem cells (BCSCs) as a kind of tumor cells are able to regenerate themselves, leading to apoptosis resistance and cancer relapse. It was reported that BCSCs contain lower levels of reactive oxygen species (ROS) associated with stemness capability. Caesalpinia sappan has been proposed for its chemopreventive potency against several cancer cells. This study aimed to evaluate the effects of Caesalpinia sappan extract (CSE) on cytotoxicity, apoptosis induction, ROS generation, and stemness markers of MDA-MB-231 and its BCSCs. 

Experimental approach:

Caesalpinia sappan was extracted under maceration with methanol. Magnetic-activated cell sorting was used to isolate BCSCs based on CD44+ and CD24- cell surface expression. The MTT test was used to assess the cytotoxic effects of CSE on MDA-MB-231 and BCSCs. Moreover, flow cytometry was used to examine the cell cycle distribution, apoptosis, ROS level, and CD44/CD24 level. Using qRT-PCR, the gene expression of the stemness markers NANOG, SOX-2, OCT-4, and c-MYC was assessed.

We found that MDA-MB-231 contains 80% of the BCSCs population, and CSE showed more potent cytotoxicity toward BCSCs than MDA-MB-231. CSE caused apoptosis in MDA-MB-231 and BCSCs cells by increasing the level of ROS. Furthermore, CSE significantly reduced the MDA-MB-231 stemness marker CD44+/CD24- and the mRNA levels of pluripotent markers of cells in BCSCs.

Conclusion and implications: 

CSE potentially reduces BCSCs stemness, which may be mediated by the elevation of the ROS levels and reduction of the expression levels of stemness transcription.

CD44 plays a pivotal role through tumorigenesis by regulating cancer cell metastasis, stemness, and chemosensitivity and is considered a promising therapeutic target for human cancers, including colorectal cancer (CRC). Therefore, the present research aimed to examine the simultaneous therapeutic effect of CD44 silencing and 5-fluorouracil (5-FU) on in vitro tumorigenesis of CRC cells.

CD44 expression was initially evaluated in TCGA datasets and CRC tissues. Furthermore, functional analysis was performed on HT-29 CRC cells overexpressing CD44. The cells were transfected with CD44 siRNA and then treated with 5-FU. Consequently, to explore the combination therapy effect on cell viability, migration, apoptosis, and chromatin fragmentation, we performed MTT assay, scratch assay, Annexin V/PI staining and DAPI staining assays, respectively. The spheroid and colony formation assays were further employed to investigate stemness features. The gene expression at protein and mRNA levels were explored using western blotting and qPCR.

Our findings illustrated that CD44 was significantly overexpressed in CRC tissues compared to normal samples. The suppression of CD44 considerably promoted the chemosensitivity of HT-29 cells to 5-FU by apoptosis induction. Also, the combination therapy led to overexpression of apoptotic genes, including P53, caspase-3, and caspase-9, as well as downregulation of AKT1 expression. Furthermore, CD44 suppression, separately or combined with 5-FU, hindered stemness properties in HT-29 cells via downregulation of Sox2 and Nanog expression. Besides, the combination therapy remarkably downregulated MMPs and suppressed CRC cell migration.

Considering its involvement in chemosensitivity to 5-FU, CD44 could be suggested as a potential target for improving the efficiency of CRC chemotherapy.

The NF-kB signaling pathway was introduced as a key pathway in carcinogenesis that is induced by inflammation in gastrointestinal malignancies. The RelA transcription factor is an important component of this signaling pathway. Furthermore, CD44 is implicated in the tumorigenesis and metastasis of gastric cancer. The aim of this study was to assay the effect of RELA knockout on CD44 expression in MKN45 cells. CRISPR/Cas9 was used to knock out RELA in MKN-45. The median fluorescence intensity (MFI) of CD44 before and after RELA knockout is analyzed in MKN45. The CRISPR/Cas9 vector pSpCas9 (BB)-2A-Puro (PX459) was used for gRNA cloning (two guides). The MKN-45 cell line was co-transfected. The purified co-transfected cells with puromycin were cultured and used for the RELA gene expression assay by real-time PCR. Flow cytometry was used for the analysis of the MFI of CD44+ in MKN45. The results showed that 180 nucleotide sequences between exon 2 and exon 3 of RELA were deleted in MKN45. RELA expression was significantly (P<0.001) decreased after CRISPR/Cas9 knockout. Compared to the control group, the MFI of CD44 in transfected cells was significantly decreased (P <0.001). Knockout of RELA significantly decreased CD44 expression in MKN45 cells. It can be concluded that the NF-kB signaling pathway via RELA is related to CD44 expression and consequently the tumorigenesis of gastric cancer. More studies about this relationship are recommended.

Expression of CD44 variant 6 (CD44v6) as a homing-associated cell adhesion molecule (HCAM),hasproved to change most cancer cells. Aim of the study is the effect of mutant allele of CD44 (rs8193C>T),Pum2regulatory element as a prognosis factor of prostate neoplasms: a case-control,in silico studies in the Mazandaranprovince-Iran.

In a case-control study,CD44-rs8193C>T genotyping of the 420 prostate neoplasms (210benign prostatic hyperplasia (BPH) patients,210 prostate cancer patients),150 healthy samples are performedby the touchdown polymerase chain reaction with confronting two-pair primers (PCR-CTPP) method. The T mutantallele effects on the mRNA structure,cell pathways were also investigated in silico methods.

Our results showed that the increase of T mutant allele frequency was significantly associated with BPHcompared with prostate cancer. Furthermore,results showed TT genotype was significantly associated with BPH[odds ratio (OR),0.572,P,0.015],and also influenced the CD44v6 transcript secondary structure,miRNA binding,and regulatory element-binding site for Pum2 protein. Attachment of Pum2 to standard CD44 transcript may lead totranscript isoform-switching,shift-expression to a variety of CD44 isoforms,which can trigger some of the cellsignaling pathways,such as Nanog-Stat,PKC-Nanog,and PKC-Twist.

Based on this,the presence of the T mutant allele of CD44 (rs8193C>T) in the populations may createa regulatory element-binding site for Pum2. So,it could be known as a prognosis factor,prediction of prostateneoplasms. However,more comprehensive studies in different populations (with various ethnicities,large populationsizes),and also CD44v6 gene expression studies in protein,transcript levels are required to confirm our data.

The clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) used for genome editing. The usage of CRISPR-Cas9 in gene editing is faced with certain limitations including off-target mutation, decreased homologous recombination (HR) repair, and immune system responses. It seems that if Cas9 expressed in an inducible manner, off-target mutations may decrease. The P53 protein decreases the activity of the HR pathway in the cell cycle, so, the decrease in P53 expression level may increase the activity of this pathway. Based on this topic, for the first time, we designed ''px601-Turbo GFP-TRE-shRNA P53'' as a CRISPR-based vector. The use of this vector can simultaneously induce expression of Cas9 and shutdown transiently P53 under an inducible promoter and an inducing agent. Therefore, shutdown transiently P53 may be leading to reduced off-targets and increased accuracy of genome editing. In the human gastric cancer MKN45 cell line, the P53 gene expresses at a normal level. Moreover, CD44 in this cell line has overexpression and is a gastric cancer stem cell marker. To evaluate this hypothesis, CD44 will be targeted for a specific sequence change (editing) by the px601-Turbo GFP-TRE-shRNA P53 vector. Accordingly, after cloning and virus preparation, MKN45 cell lines will be transduced in the presence of the appropriate doxycycline (DOX) dosage. Ultimately, to evaluate the vector efficiency, DNA extraction and whole-genome sequencing (WGS) will be done and compared with the transduced MKN45 cells without an inducible prompter and DOX as the control group. Furthermore, the Sanger sequencing for the target gene must be done. This temporary inducible expression of P53 may appear to increase the efficiency of the CD44 gene editing and reduce off-targets.

It is evident that the success of common cancer treatments is reduced due to limited drug access to tumor tissue, the drug toxicity intolerance in healthy cells, as well as the exposure of the immune system to the drug. Cancer stem cells are also a small population of tumor cells, which have different potentials for regeneration, proliferation, and differentiation and serve as a carcinogenic driving force. They are believed to play a key role in the onset, progression, drug resistance, recurrence of cancer, or metastasis. Although mesenchymal stem cells (MSC) have a slight ability to migrate toward the tumor, they could be considered as a cellular carrier for tumor targeting due to lack of recognition by the host immune system. Stem cells with their own ligands could effectively target cancer cells. One of the CD markers that exist on the surface of stem cells is CD44v6, which is considered as a homing receptor. Given that the expression level of stem cell markers is reduced during consecutive cultures in vitro environment; therefore, in the present study, stem cells were engineered using CD44 lentiviral vectors to more effectively improve the implantation and targeting of the colon cancer cell model.

In this study, the structure of the CD44 gene was designed in lentiviral vectors and transfected to the HEK293T cell line along with auxiliary plasmids PSPAX2 and PMDG2. The growth medium of virus-containing cells was collected at optimized intervals, and transduction into mice mesenchymal stem cells, injection into mice, and homing processes were traced.

Successful production of lentiviral vectors and proper expression of the corresponding factor after transduction were effective in improving the MSC homing in cancer cell.

According to these findings, it could be suggested that high expression of CD44v6 factor could be effective in improving the implantation process in cancer cells and targeting treatment.

Glioblastoma multiforme (GBM) is the most invasive type of cancer which starts inside the brain. GBM cells were found to have similar properties to glioblastoma cancer stem cells. CD44 can be used as a marker of the cancer stem cells in a subset of glioblastoma tumor cells. Recent studies showed that CD44 is involved in developing cancer cells via the protein kinase B (PKB or AKT) signaling pathway. Therefore, this study aimed to investigate the CD44 mRNA silencing effects on the glioblastoma cell cycle via AKT signaling pathway.

To determine CD44 expression in the samples of the patients with GBM, we used the analysis of data extracted from TCGA database. qRT-PCR and western blotting were used to evaluate the expression level of genes and proteins. Different cell cycles were evaluated by DAPI staining and flow cytometry.

Bioinformatics results showed that CD44 expression level in GBM tumor samples is higher than in normal samples. Effects of poly (ethylene imine)-polyethylene glycol (PEI-PEG)-loaded CD44 siRNA in cell cycle showed that CD44 silencing could inhibit cell cycle in G0-G1 phase by more than 20% compared to the negative control (P < 0.05). Furthermore, PEI-PEG-loaded CD44 siRNA reduces the expression of cyclin D1 and CKD-4. According to our findings, this structure also prevented AKT phosphorylation at Thr-308 and Ser-473.

Our results suggest that PEI-PEG-loaded CD44 siRNA may attenuate the cell cycle by suppressing AKT signaling pathway.

Cancer is a complex mechanism involving a series of cellular events. The glycoproteins such as hyaluronan (HA) are a significant element of extracellular matrix (ECM), involve in the onset of cancer developmental process. The pivotal roles of HA in cancer progression depend on dysregulated expression in various cancer. HA, also gain attention due to consideration as a primary ligand of CD44 receptor. The CD44, complex transmembrane receptor protein, due to alternative splicing in the transcription process, various CD44 isoforms predominantly exist. The overexpression of distinct CD44 isoforms (CD44v) standard (CD44s) depends on the tumour type and stage. The receptor proteins, CD44 engage in a variety of biological processes, including cell growth, apoptosis, migration, and angiogenesis. HA-CD44 interaction trigger survival pathways that result in cell proliferation, invasion ultimately complex metastasis. The interaction and binding of ligand-receptor HA-CD44 regulate the downstream cytoskeleton pathways involve in cell survival or cell death. Thus, targeting HA, CD44 (variant and standard) isoform, and HA-CD44 binding consider as an attractive and useful approach towards cancer therapeutics. The use of various inhibitors of HA, hyaluronidase (HYALs), and utilizing targeted Nano-delivery of anticancer agents and antibodies against CD44, peptides gives promising results invitro and invivo. However, they are in clinical trials with favourable and unfavourable outcomes, which reflects the need for various modifications in targeting agents and a better understanding of potential targets in tumour progression pathways.

In this study, the inhibitory effects of polyethylene imine glycol (PEI-PEG)/ CD44 siRNA nanostructures on the proliferation, invasion, and apoptosis of U87MG GBM cancer cell line, as well as the expression levels of ALDH1, RANKL, and NOTCH1 were evaluated.

In this experimental study, PEI-PEG/ CD44 siRNA nanoparticles were synthesized and characterized by atomic force microscopy (AFM), evaluation of size and zeta potential, and Fourier transform infrared (FTIR) spectroscopy. The MTT assay was adopted to evaluate the cytotoxicity of the nanoparticles. The expression levels of target genes were assessed by qRT-PCR. Flow cytometry was used for apoptosis evaluation and Trans well matrigel assay and scratch-migration were employed for investigating the invasion and migration of glioma cells.

The size and zeta potential of PEI-PEG were influenced after CD44 siRNA loading. PEI-PEG loaded with CD44 siRNAs resulted in significant inhibition of glioblastoma cell line in the concentration of 60 pmol (P<0.05). In addition, transfection of glioma cells with CD44 siRNA led to significant downregulation of ALDH1, NOTCH1, and RANKL1 (P<0.05). Transfection of this siRNA also resulted in significant suppression of invasion and migration (P<0.05).

PEI-PEG could effectively form the polyplex in combination with siRNA, be transfected into the U87MG glioma cancer cell line, and inhibit the proliferation, invasion and migration of glioma cells via suppression of ALDH1 and NOTCH1, as well as RANKL1 expression levels

Statement of the Problem:

 Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity. Early diagnosis of OSCC by using biomarkers provides preventive treatment approach to suppress the disease in early stages. CD44 as a cancer stem cell (CSC) marker may be cleaved by MT1-MMP and plays an important role in migration of cancer cells. TGF-B promotes formation of invasive cancer cells phenotype through epithelial mesenchymal transition (EMT) and induces MT1-MMP formation.

The aim of this study was to evaluate the expression of TGF-B and CD44 in leukoplakia (premalignant lesion), squamous cell carcinoma (SCC), and normal oral mucosa to determine the role of these markers in the carcinogenesis process of the oral mucosa.

The expression of TGF-B and CD44 were evaluated in 55 paraffin-embedded specimens (10normal mucosa, 15 non-dysplastic leukoplakia, 15 dysplastic leukoplakia, and 15 OSCC) by immunohistochemistry. Statistical analyses were performed using Kruskal-Wallis, Mann-Whitney, and Spearman’s rank correlation tests.

Evaluation of CD44 and TGF-B expression in the four studied groups showed statistical significant difference for each marker (p < 0.001). Pairwise comparison of CD44 and TGF-B expression in all groups except normal mucosa and non-dysplastic leukoplakia demonstrated statistical significant difference. In addition, there was positive significant correlation between two markers (r= 0.914, p < 0.001). Diagnostic test’s accuracy for identification of OSCC and dysplastic leukoplakia from non-dysplastic leukoplakia and normal tissues and recognition of OSCC from dysplastic leukoplakia showed optimum sensitivity and specificity.

Increased expression of CD44 as a cancer stem cell marker and TGF-B as an EMT marker from normal mucosa to non-dysplastic leukoplakia, dysplastic leukoplakia, and OSCC and also the significant correlation between these two markers indicated their role in carcinogenesis of oral mucosa.

The present study aimed to examine the expression of CD44 and claudin-1 markers using immunohistochemical methods to differentiate patients with minimal change disease (MCD) from those afflicted with focal segmental glomerulosclerosis (FSGS).

In this descriptive-analytical study, twenty patients with definite FSGS, twenty patients with FSGS/MCD spectrum, and seven patients with definite MCD were randomly chosen from Imam Reza hospital affiliated with Tabriz University of Medical Sciences. All patients underwent renal biopsy, and then the presence of the immune complex was examined in the obtained samples using immunofluorescence staining. Some renal specimens were paraffin-embedded for the evaluation of the expression levels of CD44 and claudin-1 utilizing the immunohistochemistry method.

Among the twenty samples obtained from patients with definite FSGS, 13 samples (65%) were double-positive for CD44 and claudin-1, 3 specimens (15%) were positive only for claudin-1, and 4 samples (20%) were double-negative for CD44 and claudin-1. The percentages of renal specimens expressing claudin-1 in patients with definite FSGS, FSGS/MCD, and MCD were 80%, 85%, and 0%, respectively. Finally, the percentages of renal samples expressing CD44 in patients with definite FSGS, FSGS/ MCD, and MCD were 65%, 10%, and 0%, respectively.

The results of the present research indicated that the rate of CD44-positive specimens was higher in patients with FSGS while the percentage of claudin-1-positive samples was more frequent in MCD patients compared with FSGS patients.