Cell aggregation activates small GTPase Rac1 and induces CD44 cleavage by maintaining lipid raft integrity.

Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell detachment from the extracellular matrix (ECM) triggers lipid raft disruption and anoikis, which is a barrier for cancer cells to metastasize. Compared to single circulating tumor cells (CTCs), our recent studies have demonstrated that CD44-mediatd cell aggregation enhances the stemness, survival and metastatic ability of aggregated cells. Here, we investigated whether and how lipid rafts are involved in CD44-mediated cell aggregation. We found that cell detachment, which mimics the condition when tumor cells detach from the ECM to metastasize, induced lipid raft disruption in single cells, but lipid raft integrity was maintained in aggregated cells. We further found that lipid raft integrity in aggregated cells was required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promoted CD44 cleavage and generated CD44 intracellular domain (CD44 ICD) to enhance stemness of aggregated cells. Consequently, lipid raft disruption inhibited Rac1 activation, CD44 ICD generation, and metastasis. Our findings reveal two new pathways regulated by CD44-mediated cell aggregation via maintaining lipid raft integrity. These findings also suggest that targeting cell aggregation-mediated pathways could be a novel therapeutic strategy to prevent CTC cluster-initiated metastasis.

High prevalence of CD44 and its ligand low molecular weight hyaluronan in plasma of HNSCC patients: clinical significance.

BACKGROUND: This study aims to evaluate the role of cancer stem cell marker, CD44, and its ligand HA as potential molecular biomarker for early detection of HNSCC. METHODS AND RESULTS: The expression profile (mRNA/Protein) of CD44 variants were analysed in primary HNSCC lesions and plasma of the patients. Then, prevalence of HA variants was analysed in plasma of the patients. The mRNA expression of CD44 variants, CD44S and CD44v3, were significantly high in both early (stage I/II) and late (stage III/IV) invasive lesions, with predominant expression of CD44v3 in the late-stage lesions. In plasma of HNSCC patients, increased levels of SolCD44, CD44-ICD and unique 62 KD CD44 variants with respect to standard CD44S were seen, in comparison to their prevalence in plasma of normal individuals. The abundance of CD44-ICD and 62 KD variants were significantly high in plasma of late stage HNSCC patients. Interestingly, significantly high level of low molecular weight HA(LMW HA) with respect to high molecular weight HA(HMW HA) was seen in plasma of HNSCC patients irrespective of clinical stages. On the contrary, high HMW HA level in plasma of normal individuals was seen. The high level of LMW HA in plasma of HNSCC patients might be due to combinatorial effect of increased mRNA expression of HA synthesizing enzyme HAS1/2/3 and HA degrading enzyme HYAL1/2, as seen in the primary HNSCC samples. CONCLUSION: Thus, our data revealed the importance of specific CD44 and HA variants in plasma of HNSCC patients during its development as potential non-invasive molecular biomarker of the disease.

Inhibiting CD44-ICD Attenuates LPS-Induced Initiation of Hepatic Inflammation in Septic Mice.

Sepsis is a severe condition induced by microbial infection. It elicits a systemic inflammatory response, leading to multi-organ failure, and the liver, as a scavenger, plays a significant role in this process. Controlling hepatic inflammation and maintaining liver function is crucial in managing sepsis. CD44-ICD, as a CD44 signal transductor, is involved in multiple inflammatory responses. However, the role of CD44-ICD in lipopolysaccharide (LPS)-induced hepatic inflammation has not been investigated. Therefore, we aimed to examine whether CD44-ICD initiates hepatic inflammation in septic mice. We induced hepatic inflammation in mice by administering LPS. DAPT, a CD44-ICD inhibitor, was given to mice or Chang cells 30 min or 1 h before LPS administration (10 mg/kg, i.p., or 100 ng/mL, respectively). Inhibition of CD44-ICD decreased the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), hepatic necrosis, inflammatory cell infiltration, interleukin (IL)-1ß, inducible NO synthase (iNOS), nitric oxide (NO) production, nuclear factor (NF)κB signaling pathway proteins, and CD44 expression in mice. CD44-ICD inhibition also decreased IL-1ß and CD44 expression levels in Chang cells. CD44-ICD may be a primary regulatory function in CD44-associated LPS-induced initiation of hepatic inflammation in mice.

Characterization of CD44 intracellular domain interaction with RUNX2 in PC3 human prostate cancer cells.

BACKGROUND: Expression of CD44 receptor is associated with the onset of several tumors. The intracellular domain of CD44 (CD44-ICD) has been implicated as a co-transcription factor for RUNX2 in the regulation of expression of MMP-9 in breast carcinoma cells. Previous studies from our laboratory demonstrated the role of CD44 in migration and invasion of PC3 prostate cells through activation of MMP-9. CD44 signaling regulates the phosphorylation and hence the localization of RUNX2 in the nucleus. The role of CD44-ICD has not been studied in prostate cancer cells. This study aimed to explore the role of CD44-ICD and RUNX2 in the regulation of expression of metastasis-related genes. METHODS: PC3 and PC3 cells overexpressing RUNX2 protein were analyzed for RUNX2/CD44-ICD interaction by immunoprecipitation, immunoblotting, and Immunofluorescence analyses. Wound healing and tumorsphere formation analyses were also done in these cells. The real-time PCR analysis was used to detect the expression levels of different genes. RESULTS: Expression of CD44 and RUNX2 was observed only in PC3 cells (androgen receptor positive) and not in LNCaP or PCa2b cells (androgen receptor negative). Therefore, CD44-ICD fragment (~ 15-16 kDa) was observed in PC3 cells. Moreover, localization of CD44-ICD was more in the nucleus than in the cytoplasm of PC3 cells. Inhibition of cleavage of CD44 with a γ-secretase inhibitor, DAPT reduced the formation of CD44-ICD; however, accumulation of CD44-external truncation fragments (~ 20 and ~ 25 kDa) was detected. RUNX2 and CD44-ICD interact in the nucleus of PC3 cells, and this interaction was more in PC3 cells transfected with RUNX2 cDNA. Overexpression of RUNX2 augments the expression of metastasis-related genes (e.g., MMP-9 and osteopontin) which resulted in increased migration and tumorsphere formation. CONCLUSIONS: We have shown here a strong functional relationship between CD44-ICD and RUNX2 in PC3 cells. RUNX2 forms a complex with CD44-ICD as a co-transcriptional factor, and this complex formation not only activates the expression of metastasis-related genes but also contributes to migration and tumorsphere formation. Therefore, RUNX2 and CD44-ICD are potential targets for anti-cancer therapy, and attenuation of their interaction may validate the regulatory effects of these proteins on cancer migration and progression.

CD44 In Sarcomas: A Comprehensive Review and Future Perspectives.

It is widely accepted that the tumor microenvironment, particularly the extracellular matrix, plays an essential role in the development of tumors through the interaction with specific protein-membrane receptors. One of the most relevant proteins in this context is the transmembrane protein CD44. The role of CD44 in tumor progression, invasion, and metastasis has been well established in many cancers, although a comprehensive review concerning its role in sarcomas has not been published. CD44 is overexpressed in most sarcomas and several in vitro and in vivo experiments have shown a direct effect on tumor progression, dissemination, and drug resistance. Moreover, CD44 has been revealed as a useful marker for prognostic and diagnostic (CD44v6 isoform) in osteosarcoma. Besides, some innovative treatments such as HA-functionalized liposomes therapy have become an excellent CD44-mediated intracellular delivery system for osteosarcoma. Unfortunately, the reduced number of studies deciphering the prognostic/diagnostic value of CD44 in other sarcoma subgroups, neither than osteosarcoma, in addition to the low number of patients involved in those studies, have produced inconclusive results. In this review, we have gone through the information available on the role of CD44 in the development, maintenance, and progression of sarcomas, analyzing their implications at the prognostic, therapeutic, and mechanistic levels. Moreover, we illustrate how research involving the specific role of CD44 in the different sarcoma subgroups could suppose a chance to advance towards a more innovative perspective for novel therapies and future clinical trials.

Reg4 and its downstream transcriptional activator CD44ICD in stage II and III colorectal cancer.

Reg4 is highly expressed in gastrointestinal malignancies and acts as a mitogenic and pro-invasive factor. Our recent works suggest that Reg4 binds with CD44 and induces its proteolytic cleavage to release intra-cytoplasmic domain of CD44 (CD44ICD). The goal of this study is to demonstrate clinical significance of the Reg4-CD44/CD44ICD pathway in stage II/III colon cancer and its association with clinical parameters of aggression. We constructed a tissue microarray (TMA) of 93 stage II/III matched colon adenocarcinoma patients, 23 with recurrent disease. The TMA was immunohistochemically stained for Reg4, CD44, and CD44ICD proteins and analyzed to identify associations with tumor characteristics, recurrence and overall survival. The TMA data analysis showed a significant correlation between Reg4 and CD44 (r2 = 0.23, P = 0.028), CD44 and CD44ICD (r2 = 0.36, p = 0.0004), and Reg4 and CD44ICD (r2 = 0.45, p ≤ 0.0001). Reg4 expression was associated with larger tumor size (r2 = 0.23, p = 0.026). Although, no association was observed between Reg4, CD44, or CD44ICD expression and disease recurrence, Reg4-positive patients had a median survival of 4 years vs. 7 years for Reg4-negative patients (p = 0.04) in patients who recurred. Inhibition of the Reg4-CD44/CD44ICD pathway may be a future therapeutic target for colon cancer patients.

High molecular weight form of hyaluronic acid reduces neuroinflammatory response in injured sciatic nerve via the intracellular domain of CD44.

Inflammatory response after peripheral nerve injury is required for clearance of tissue debris and effective regeneration. Studies have revealed that hyaluronic acid (HA) may exert different properties depending on their molecular size. High molecular weight HA (>>1,000 kDa; HMW-HA) displays immunosuppressive properties, whereas low molecular weight HA (<800 kDa; LMW-HA) induces proinflammatory responses. The role of HMW-HA interaction with CD44, a major HA receptor, in neuroinflammatory responses has not been fully elucidated. The purpose of this experimental study was to investigate the effects of topical applications of HMW-HA on the sciatic nerve injury in an adult rat model. At the crush site on the sciatic nerve, the recordings of compound muscle action potential (CMAP) and the levels of several proteins related to inflammatory response were assessed at time intervals of 2, 4, and 6 weeks postsurgery. Here, we show that the recovery effect of HMW-HA treatment had significantly shortened latency and increased amplitude of CMAP compared with crushed alone, crushed plus γ-secretase inhibitor with or without HA treatment at 6 weeks after surgery. Our data reveal that HMW-HA could downregulate the expression of IL1-ß, TLR4, and MMP-9, whereas these proteins expression were increased when the CD44-ICD activity was inhibited using γ-secretase inhibitor. Our findings demonstrated a novel role of CD44-ICD in HA-mediated recovery of peripheral nerve injury. Clinical relevance: an alternative for the regeneration of peripheral nerve injury.

Identification of sequence-specific interactions of the CD44-intracellular domain with RUNX2 in the transcription of matrix metalloprotease-9 in human prostate cancer cells.

AIM: The Cluster of differentiation 44 (CD44) transmembrane protein is cleaved by γ-secretase, the inhibition of which blocks CD44 cleavage. This study aimed to determine the biological consequence of CD44 cleavage and its potential interaction with Runt-related transcription factor (RUNX2) in a sequence-specific manner in PC3 prostate cancer cells. METHODS: Using full-length and C-terminal deletion constructs of CD44-ICD (D1-D5) expressed as stable green fluorescent protein-fusion proteins in PC3 cells, we located possible RUNX2-binding sequences. RESULTS: Chromatin immunoprecipitation assays demonstrated that the C-terminal amino acid residues between amino acids 671 and 706 in D1 to D3 constructs were indispensable for sequence-specific binding of RUNX2. This binding was minimal for sequences in the D4 and D5 constructs. Correspondingly, an increase in matrix metalloprotease-9 (MMP-9) expression was observed at the mRNA and protein levels in PC3 cells stably expressing D1-D3 constructs. CONCLUSION: These results provide biochemical evidence for the possible sequence-specific CD44-ICD/RUNX2 interaction and its functional relationship to MMP-9 transcription in the promoter region.

CD44ICD promotes breast cancer stemness via PFKFB4-mediated glucose metabolism.

CD44 is a single-pass cell surface glycoprotein that is distinguished as the first molecule used to identify cancer stem cells in solid tumors based on its expression. In this regard, the CD44high cell population demonstrates not only the ability to regenerate a heterogeneous tumor, but also the ability to self-regenerate when transplanted into immune-deficient mice. However, the exact role of CD44 in cancer stem cells remains unclear in part because CD44 exists in various isoforms due to alternative splicing. Methods: Gain- and loss-of-function methods in different models were used to investigate the effects of CD44 on breast cancer stemness. Cancer stemness was analyzed by detecting SOX2, OCT4 and NANOG expression, ALDH activity, side population (SP) and sphere formation. Glucose consumption, lactate secretion and reactive oxygen species (ROS) levels were detected to assess glycolysis. Western blot, immunohistochemical staining, ELISA and TCGA dataset analysis were performed to determine the association of CD44ICD and PFKFB4 with clinical cases. A PFKFB4 inhibitor, 5MPN, was used in a xenograft model to inhibit breast cancer development. Results: In this report, we found that the shortest CD44 isoform (CD44s) inhibits breast cancer stemness, whereas the cleaved product of CD44 (CD44ICD) promotes breast cancer stemness. Furthermore, CD44ICD interacts with CREB and binds to the promoter region of PFKFB4, thereby regulating PFKFB4 transcription and expression. The resultant PFKFB4 expression facilitates the glycolysis pathway (vis-à-vis oxidative phosphorylation) and promotes stemness of breast cancer. In addition, we found that CD44ICD and PFKFB4 expressions are generally up-regulated in the tumor portion of breast cancer patient samples. Most importantly, we found that 5MPN (a selective inhibitor of PFKFB4) suppresses CD44ICD-induced tumor development. Conclusion: CD44ICD promotes breast cancer stemness via PFKFB4-mediated glycolysis, and therapies that target PFKFB4 (e.g., 5MPN therapy) may lead to improved outcomes for cancer patients.

CD44: A Multifunctional Cell Surface Adhesion Receptor Is a Regulator of Progression and Metastasis of Cancer Cells.

CD44 is a cell surface adhesion receptor that is highly expressed in many cancers and regulates metastasis via recruitment of CD44 to the cell surface. Its interaction with appropriate extracellular matrix ligands promotes the migration and invasion processes involved in metastases. It was originally identified as a receptor for hyaluronan or hyaluronic acid and later to several other ligands including, osteopontin (OPN), collagens, and matrix metalloproteinases. CD44 has also been identified as a marker for stem cells of several types. Beside standard CD44 (sCD44), variant (vCD44) isoforms of CD44 have been shown to be created by alternate splicing of the mRNA in several cancer. Addition of new exons into the extracellular domain near the transmembrane of sCD44 increases the tendency for expressing larger size vCD44 isoforms. Expression of certain vCD44 isoforms was linked with progression and metastasis of cancer cells as well as patient prognosis. The expression of CD44 isoforms can be correlated with tumor subtypes and be a marker of cancer stem cells. CD44 cleavage, shedding, and elevated levels of soluble CD44 in the serum of patients is a marker of tumor burden and metastasis in several cancers including colon and gastric cancer. Recent observations have shown that CD44 intracellular domain (CD44-ICD) is related to the metastatic potential of breast cancer cells. However, the underlying mechanisms need further elucidation.