Expression Of CD44 Variants Research Articles

Anti-CD44 Variant 10 Monoclonal Antibody Exerts Antitumor Activity in Mouse Xenograft Models of Oral Squamous Cell Carcinomas.

CD44 regulates cell adhesion, proliferation, survival, and stemness and has been considered a tumor therapy target. CD44 possesses the shortest CD44 standard (CD44s) and a variety of CD44 variant (CD44v) isoforms. Since the expression of CD44v is restricted in epithelial cells and carcinomas compared to CD44s, CD44v has been considered a promising target for monoclonal antibody (mAb) therapy. We previously developed an anti-CD44v10 mAb, C44Mab-18 (IgM, kappa), to recognize the variant exon 10-encoded region. In the present study, a mouse IgG2a version of C44Mab-18 (C44Mab-18-mG2a) was generated to evaluate the antitumor activities against CD44-positive cells compared with the previously established anti-pan CD44 mAb, C44Mab-46-mG2a. C44Mab-18-mG2a exhibited higher reactivity compared with C44Mab-46-mG2a to CD44v3-10-overexpressed CHO-K1 (CHO/CD44v3-10) and oral squamous cell carcinoma cell lines (HSC-2 and SAS) in flow cytometry. C44Mab-18-mG2a exerted a superior antibody-dependent cellular cytotoxicity (ADCC) against CHO/CD44v3-10. In contrast, C44Mab-46-mG2a showed a superior complement-dependent cytotoxicity (CDC) against CHO/CD44v3-10. A similar tendency was observed in ADCC and CDC against HSC-2 and SAS. Furthermore, administering C44Mab-18-mG2a or C44Mab-46-mG2a significantly suppressed CHO/CD44v3-10, HSC-2, and SAS xenograft tumor growth compared with the control mouse IgG2a. These results indicate that C44Mab-18-mG2a could be a promising therapeutic regimen for CD44v10-positive tumors.

Molecular biological role of epithelial splicing regulatory protein1 in intrahepatic cholangiocarcinoma.

Epithelial splicing regulatory protein1 (ESRP1) regulates tumor progression and metastasis through the epithelial‒mesenchymal transition by interacting with zinc finger E-box binding1 (ZEB1) and CD44 in cancers. However, the role of ESRP1 in intrahepatic cholangiocarcinoma (iCCA) remains unclear. Three iCCA cell lines (HuCCT-1, SSP-25, and KKU-100) were analyzed using small interfering RNA to investigate the molecular biological functions of ESRP1 and ZEB1. The association between clinicopathological features and the expression of ESRP1 and ZEB1 in iCCA tissues was analyzed immunohistochemically. Proteomic analysis was performed to identify molecules related to ESRP1 expression. ESRP1 expression was upregulated in HuCCT-1 and SSP-25 cells. Cell migration and invasion were enhanced, and the expression of ZEB1 and CD44s (CD44 standard) isoforms were upregulated in the ESRP1 silencing cells. Moreover, ESRP1 silencing increased the expression of N-cadherin and vimentin, indicating the presence of mesenchymal properties. Conversely, ZEB1 silencing increased the expression of ESRP1 and CD44v (CD44 variant) isoforms. Immunohistochemical analysis revealed that a lower ESRP1-to-ZEB1 expression ratio was associated with poor recurrence-free survival in patients with iCCA. Flotillin 2, a lipid raft marker related to epithelial‒mesenchymal transition, was identified as a protein related to the interactive feedback loop in proteomic analysis. ESRP1 suppresses tumor progression in iCCA by interacting with ZEB1 and CD44 to regulate epithelial‒mesenchymal transition.

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

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. 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. 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.

Late recurrent gastric carcinoma 12 years after surgery with attenuation of CD44 variant 9 expression

BackgroundLate recurrence of gastric cancer at 10 years post-gastrectomy is extremely rare, and the underlying mechanism remains unclear. We report a para-aortic lymph node metastasis case that recurred 12 years postoperatively.Case presentationA 44-year-old woman pathologically diagnosed with moderately to poorly differentiated adenocarcinoma with pT2(SS)pN2cM0pStageIIIA according to the Japanese Classification of Gastric Carcinoma (the 13th Edition) underwent laparoscopic distal gastrectomy with D1 + lymph node dissection. She received adjuvant chemotherapy with tegafur-uracil (400 mg/day) for 2 years. At postoperative year (POY) 5, a swollen lymph node was detected in the No.16b1lat lymph node station. However, positron emission tomography (PET) revealed normal uptake, and the levels of tumor markers were within normal limits; hence, the possibility of metastasis was considered low, and the patient was placed under observation. At POY 12, computed tomography revealed an enlargement of the No.16b1lat lymph node station, and PET showed abnormal uptake. Endoscopic ultrasound-guided fine-needle aspiration revealed a moderately differentiated adenocarcinoma. Hence, a diagnosis of recurrence of gastric cancer was made. The patient underwent para-aortic nodal dissection (PAND) of No.16b1lat & int stations. Immunochemical staining results also suggested the recurrence of gastric cancer. However, the expression of CD44 variant 9 (CD44v9), a cancer stem cell marker for gastric adenocarcinoma, was attenuated in the recurrent lesions compared with that in the primary lesions. Postoperatively, she received chemotherapy with tegafur–gimeracil–oteracil (80 mg/day) for 1 year. Bone metastasis was observed at POY 4 after PAND, and the IHC analysis showed a HER2 score of 3 + in a needle biopsy specimen of bone metastasis. The expression of CD44v9 was slightly positive. The patient is being treated with chemotherapy with FOLFOX + trastuzumab.ConclusionsA defense mechanism against reactive oxygen species has been reported as a mechanism causing recurrence of CD44v9-positive gastric cancer. Consequently, CD44v9-positive gastric cancer grows in metastatic organs, repeatedly self-renews, and proliferates to form recurrent lesions. In the present case, the degree of CD44v9 staining in recurrent lesions was suggested to be related to the recurrence time.

Targeting CD44 Variant 5 with an Antibody-Drug Conjugate Is an Effective Therapeutic Strategy for Intrahepatic Cholangiocarcinoma.

Elevated expression of CD44 variant 5 in intrahepatic cholangiocarcinoma confers a targetable vulnerability using the newly developed antibody-drug conjugate H1D8-DC, which induces potent growth suppressive effects without significant toxicity.

Mutant Allele of CD44 (rs8193C>T) and Pum2 Regulatory Element as A Prognosis Factor of Prostate Neoplasms: A Case-Control and In Silico Studies.

Expression of CD44 variant 6 (CD44v6) as a homing-associated cell adhesion molecule (HCAM), has proved to change most cancer cells. Aim of the study is the effect of mutant allele of CD44 (rs8193C>T) and Pum2 regulatory element as a prognosis factor of prostate neoplasms: a case-control and in silico studies in the Mazandaran province-Iran. In a case-control study, CD44-rs8193C>T genotyping of the 420 prostate neoplasms (210 benign prostatic hyperplasia (BPH) patients and 210 prostate cancer patients) and 150 healthy samples are performed by the touchdown polymerase chain reaction with confronting two-pair primers (PCR-CTPP) method. The T mutant allele effects on the mRNA structure and cell pathways were also investigated in silico methods. Our results showed that the increase of T mutant allele frequency was significantly associated with BPH compared with prostate cancer. Furthermore, results showed TT genotype was significantly associated with BPH [odds ratio (OR)=0.572 and 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 to transcript isoform-switching and shift-expression to a variety of CD44 isoforms, which can trigger some of the cell signaling 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 create a regulatory element-binding site for Pum2. So, it could be known as a prognosis factor and prediction of prostate neoplasms. However, more comprehensive studies in different populations (with various ethnicities and large population sizes), and also CD44v6 gene expression studies in protein and transcript levels are required to confirm our data.

Anti-inflammatory effects of differential molecular weight Hyaluronic acids on UVB-induced calprotectin-mediated keratinocyte inflammation

BackgroundThe biological functions of Hyaluronic acid are related to its molecular weight and binding to its receptor, Toll-like receptor4 (TLR4) or CD44. Recent studies have shown that low-molecular-weight Hyaluronic acid (LMW-HA) exhibits proinflammatory effects, while high-molecular-weight Hyaluronic acid (HMW-HA) functions as an anti-inflammatory factor. UVB-induced epidermal inflammation is mainly mediated by endogenous molecules, such as damage-associated molecular patterns (DAMPs), that cause severe skin damage by activating TLR signaling pathways. ObjectiveSince both LMW- and HMW-HA have inhibitory functions on TLR-mediated macrophage inflammation, HA is assumed to suppress UVB-induced DAMP-mediated inflammation in the skin. In this study, both Ultra- low-molecular-weight Hyaluronic acid (uLMW-HA) and HMW-HA were found to inhibit UVB-induced keratinocyte inflammation. MethodsHaCaT cells were treated with medium containing Hyaluronic acid at the appropriate concentration after 15 mJ/cm2 irradiation. Secreted protein levels were determined with ELISA kits. Expression levels of proteins downstream of TLR4 were detected by Simple Western system. ResultsBy competitively binding to TLR4, uLMW-HA downregulated Calprotectin-induced TRAF6 expression, which might be the direct process by which uLMW-HA decreased UVB-induced IL-6 secretion. Reduced CD44 variant (CD44v) expression in keratinocytes attenuated the inhibitory effect of both uLMW-HA and HMW-HA on UVB-induced inflammation, which indicated the involvement of CD44v in HA-regulated anti-inflammatory activity. ConclusionOverall, this research indicates that Hyaluronic acid is more than a moisturizer; it is also a biologically effective material that can prevent the excessive skin inflammation caused in daily life, especially in the late stages after sunburn.

The upregulation of circFNDC3B aggravates the recurrence after endoscopic submucosal dissection (ESD) in early gastric cancer (EGC) patients

It has been reported that the expression of CD44 variant 9 could be utilized as a predictive marker for the recurrence in early gastric cancer (EGC) after endoscopic submucosal dissection (ESD). And circFNDC3B was proved to increase the migration and invasion of gastric cancer (GC) cells. In this study, we recruited 96 EGC patients after ESD treatment and grouped them into High circFNDC3B expression group (High expression group) and Low circFNDC3B expression group (Low expression group). Accordingly, we found that the recurrence-free rate in the High expression group was lower than that in the Low expression group. In the High expression group, the relative expression of miR-942 and miR-510 was both suppressed while the relative expression of CDH1 mRNA and CD44 mRNA/protein was increased compared with those in the Low expression group. CircFNDC3B was found to target miR-942 and miR-510 and suppress their expressions respectively. Moreover, miR-942 was found to target CD44 mRNA while miR-510 was found to target CDH1 mRNA. The overexpression of circFNDC3B led to the down-regulation of miR-942 and miR-510, which accordingly resulted in the up-regulation of CD44 and CDH1 in MKN28 cells. Moreover, we found H. pylori infection could promote the expression of circFNDC3B, which also resulted in up-regulated CD44 and CDH1 mRNA level in rTip-α cultivated MKN28 cells. In summary, our study demonstrated that a higher level of circFNDC3B could lead to the increased expression of CD44 and CDH1 via modulating the signaling pathways of miR-942/CD44 and miR-510/CDH1 in EGC patients. And the up-regulation of CD44 and CDH1 would accordingly result in a higher recurrence rate of EGC patients treated by ESD.

Multispectral LEDs Eliminate Lipofuscin-Associated Autofluorescence for Immunohistochemistry and CD44 Variant Detection by in Situ Hybridization in Aging Human, non-Human Primate, and Murine Brain.

A major limitation of mechanistic studies in aging brains is the lack of routine methods to robustly visualize and discriminate the cellular distribution of tissue antigens using fluorescent immunohistochemical multi-labeling techniques. Although such approaches are routine in non-aging brains, they are not consistently feasible in the aging brain due to the progressive accumulation of autofluorescent pigments, particularly lipofuscin, which strongly excite and emit over a broad spectral range. Consequently, aging research has relied upon colorimetric antibody techniques, where discrimination of tissue antigens is often challenging. We report the application of a simple, reproducible, and affordable protocol using multispectral light-emitting diodes (mLEDs) exposure for the reduction/elimination of lipofuscin autofluorescence (LAF) in aging brain tissue from humans, non-human primates, and mice. The mLEDs lamp has a broad spectral range that spans from the UV to infrared range and includes spectra in the violet/blue and orange/red. After photo quenching, the LAF level was markedly reduced when the tissue background fluorescence before and after mLEDs exposure was compared (p < 0.0001) across the spectral range. LAF elimination was estimated at 95 ± 1%. This approach permitted robust specific fluorescent immunohistochemical co-visualization of commonly studied antigens in aging brains. We also successfully applied this method to specifically visualize CD44 variant expression in aging human cerebral white matter using RNAscope fluorescent in-situ hybridization. Photo quenching provides an attractive means to accelerate progress in aging research by increasing the number of molecules that can be topologically discriminated by fluorescence detection in brain tissue from normative or pathological aging.

Tumor-Infiltrating Lymphocytes and Cancer Markers in Osteosarcoma: Influence on Patient Survival.

Simple SummaryOsteosarcoma (OST) is the most common type of high-grade primary bone tumor, which mainly affects young adults. Despite the efforts that have been made to address the importance of immune-related factors in OST, there is still a lot to understand. The purpose of the current study was to evaluate the tumor-infiltrating lymphocytes (TIL), the expression of proteins involved in tumor biology, and their impact on the clinical outcome of OST patients. Our results suggest that the presence of tumor-infiltrating CD4+ cells provides protection to patients, and that CD8+ cells have a significant impact on the patient’s overall survival (OS) and progression-free survival (PFS). In addition, a strong association of tumor-infiltrating CD4+ cells and the presence of CD44s expression in tumor samples was observed. These findings reinforce the idea that TIL and the expression of tumor markers should be taken into consideration in order to improve OST treatment and management.Osteosarcoma (OST) is the most common type of high-grade primary bone tumor, which mainly affects young adults. The current standard of care for OST combines surgical resection with chemotherapy. The clinical outcomes and the current options to treat OST patients are unsatisfactory and novel treatment strategies are needed. The crosstalk between tumor cells and immune cells is essential to the OST microenvironment. Despite the efforts that have been made to address the importance of immune-related factors in OST, there is still a lot to understand. The purpose of the current study was to evaluate the tumor-infiltrating lymphocytes (TIL), the expression of proteins involved in tumor biology, and their impact on the clinical outcome of OST patients. We studied 93 samples of OST patients using immunohistochemistry and histomorphometry. We looked for the infiltration of CD3+, CD4+, CD8+, TIA1+ and CD20+ cells and for the expression of CD44 standard (CD44s) and variant 6 (CD44v6), CD95/Fas, Fas-L, p53 and p-glycoprotein. All the parameters were analyzed for the influence on the occurrence of death and metastasis, plus patient overall survival (OS) and progression-free survival (PFS). The effect of sex, age, tumor location (distal femur or proximal tibia) and the combination with neoadjuvant chemotherapy was also assessed. Our results suggest that the presence of tumor-infiltrating CD4+ cells provides protection to OST patients, and that CD8+ cells have a significant impact on the patient’s overall survival (OS) and progression-free survival (PFS), which is more evident in male patients. In addition, a strong association between tumor-infiltrating CD4+ cells and the presence of CD44s expression in tumor samples was observed. Analysis of TIL and tumor markers related to tumor biology could be useful to stratify patients and monitor the response to therapy, as well as to assist with the development of immunotherapy strategies to improve the effects of cytotoxic TIL to eradicate the tumor cells.

Deciphering the Molecular Machinery-Influence of sE-Cadherin on Tumorigenic Traits of Prostate Cancer Cells.

Simple SummaryDespite recent advances in the therapeutic management of metastasized prostate cancer, disease progression is still inevitable, with often fatal outcomes. Elucidating molecular mechanisms crucial to cancer development and progression is therefore necessary to find ways to interfere in metastatic processes and ultimately improve prognosis. Since soluble (s)E-cadherin is elevated in the serum of patients with prostate cancer, we investigated its influence on prostate cancer cell behavior in vitro. Exposure to sE-cadherin increased the systemic spread of the cells. Thus, targeting sE-cadherin might be a novel and innovative concept to treat advanced PCa.The serum level of soluble (s)E-cadherin is elevated in several malignancies, including prostate cancer (PCa). This study was designed to investigate the effects of sE-cadherin on the behavior of PCa cells in vitro, with the aim of identifying a potential therapeutic target. Growth as well as adhesive and motile behavior were evaluated in PC3, DU-145, and LNCaP cells. Flow cytometry was used to assess cell cycle phases and the surface expression of CD44 variants as well as α and β integrins. Confocal microscopy was utilized to visualize the distribution of CD44 variants within the cells. Western blot was applied to investigate expression of α3 and β1 integrins as well as cytoskeletal and adhesion proteins. Cell growth was significantly inhibited after exposure to 5 µg/mL sE-cadherin and was accompanied by a G0/G1-phase arrest. Adhesion of cells to collagen and fibronectin was mitigated, while motility was augmented. CD44v4, v5, and v7 expression was elevated while α3 and β1 integrins were attenuated. Blocking integrin α3 reduced cell growth and adhesion to collagen but increased motility. sE-cadherin therefore appears to foster invasive tumor cell behavior, and targeting it might serve as a novel and innovative concept to treat advanced PCa.

Correction to: Expression of CD44 variant 9 induces chemoresistance of gastric cancer by controlling intracellular reactive oxygen spices accumulation

Correction to: Expression of CD44 variant 9 induces chemoresistance of gastric cancer by controlling intracellular reactive oxygen spices accumulation

Expression of CD44 variant 9 induces chemoresistance of gastric cancer by controlling intracellular reactive oxygen spices accumulation.

CD44 variant 9 (CD44v9) has been reported to suppress reactive oxygen spices (ROS) in association with antioxidant factors such as glutathione (GSH) and glutathione peroxidase 2 (GPx2), resulting in promoted tumor growth. CD44v9 and GPx2 expression were investigated by immunohistochemistry in resected specimens from 193 gastric cancer (GC) patients without preoperative chemotherapy and in pretreatment biopsy specimens from 29 GC patients with preoperative chemotherapy. We analyzed the relationship between CD44v9 expression and clinicopathological factors, prognosis, and pathological response to chemotherapy. In GC cell lines, we examined the relationship between CD44v9 expression and chemotherapeutic sensitivity. In patients without preoperative chemotherapy, CD44v9 expression was significantly associated with depth of invasion, lymphatic permeation, vascular invasion, distant metastasis and GPx2 expression. In multivariate analysis, CD44v9 expression was an independent poor prognosis factor for overall survival and recurrence-free survival. In patients with preoperative chemotherapy, CD44v9 expression was significantly associated with worse pathological response and GPx2 expression. In GC cell lines, downregulation of CD44v9 expression enhanced chemotherapeutic sensitivity to 5-fluorouracil with changing GSH and ROS levels. CD44v9-positive expression was associated with chemotherapeutic resistance by controlling intracellular accumulated ROS, suggesting that CD44v9 may be a predictive biomarker for chemotherapy in GC.

KHDRBS3 promotes multi-drug resistance and anchorage-independent growth in colorectal cancer.

5‐Fluorouracil (5‐FU) is one of the most frequently used pharmacological agents in the treatment of colorectal cancer (CRC). Resistance to chemotherapy is a major cause of treatment failure of CRC, and it is a well known fact that cancer stem cells play a significant role in the acquisition of drug resistance. In this study, we focused on the KHDRBS3 gene that encodes KH RNA Binding Domain Containing, Signal Transduction Associated 3. We first clarified the relationship between KHDRBS3 and 5‐FU resistance. We then observed higher expression levels of KHDRBS3 in KRAS‐mutant organoids and cell lines in comparison with KRAS wild‐type organoids and cell lines. Immunohistochemical analysis using CRC cases revealed that the prognosis of KHDRBS3‐positive patients was significantly worse compared with that of KHDRBS3‐negative patients. Univariate and multivariate Cox proportional hazards analyses showed that KHDRBS3 was an independent prognostic factor in patients with CRC. We determined that KHDRBS3 might play a crucial role in the acquisition of stem cell properties, such as drug resistance and spheroid/organoid formation, by regulating CD44 variant expression and the Wnt signaling pathway. In an immunodeficient mouse model, KHDRBS3‐positive cells showed efficient tumor formation and formed metastatic lesions in the lungs. These results indicated that KHDRBS3 plays a crucial role in drug resistance and anchorage‐independent growth by maintaining stem cell‐like features in CRC cells. KHDRBS3 could be a promising candidate marker for predicting chemotherapeutic effect and prognosis in CRC patients.

Functional analysis of CD44 variants and xCT in canine tumours.

The cell surface glycoprotein CD44 has various types of splicing variants, which contribute to its multiple distinct cellular functions. Recently, it was reported that the CD44v8‐10 isoform interacts with the system Xc(‐) transporter‐related protein (xCT), and inhibits the accumulation of reactive oxygen species by promoting the synthesis of the antioxidant glutathione in human tumour cells. In this study, we investigated the expression and function of CD44 variants and xCT in canine tumours. From semi‐quantitative reverse transcription polymerase chain reaction analysis, the mRNA expression of the CD44v8‐10 isoform was observed in canine tumour tissues as well as human cases. The overexpression of CD44v8‐10 may promote the synthesis of glutathione and enhance the resistance to radiation of canine breast tumour cells. Furthermore, canine xCT mRNA expression was significantly upregulated in the canine breast tumour tissues as compared to the normal tissues surrounding the tumours. To investigate the function of canine xCT, we treated canine tumour cells with the xCT inhibitor sulfasalazine. Consequently, the sulfasalazine‐treated cells were more sensitive to oxidative stress than the non‐treated cells. Taken together, these results suggested that CD44v8‐10 and xCT play important roles in the therapy resistance of canine tumours as well as human tumours.

Molecular biological analysis of 5-FU-resistant gastric cancer organoids; KHDRBS3 contributes to the attainment of features of cancer stem cell.

5-FU is one of the key drugs in the treatment of gastric cancer (GC). Much evidence has shown that cancer stem cells (CSCs) play a key role in the acquisition of drug resistance. The organoid is a novel 3D cell culture system technology that sustains stem-cell-driven formation of near-physiological, self-renewing tissues using specific niche factors in a dish. In this study, we established GC organoids (GCOs) and gradually treated them with higher concentrations of 5-FU. We successfully harvested four 5-FU-resistant GCOs, which were supported by significant changes in the expression of molecules related to 5-FU metabolism. We then performed microarray analysis using three normal gastric organoids and three pairs of 5-FU-resistant and parental GCOs. Through the comparison of expression profiles and further validation, we chose KHDRBS3 as a target gene. We found KHDRBS3 to be an independent prognostic factor in GC patients, especially in GC patients treated with 5-FU chemotherapy. We also determined that KHDRBS3 might play an important role in the acquisition of stem cell-like features, such as multi-drug resistance and organoid formation, by regulating CD44 variant expression. We found KHDRBS3, which is thought to play an important role in the acquisition of characteristics of CSCs in GC, to be a promising candidate marker for predicting therapeutic effect and prognosis in GC patients.

Endothelial-Mesenchymal Transition Drives Expression of CD44 Variant and xCT in Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) pathogenesis shares similarities with carcinogenesis. One CD44 variant (CD44v) isoform, CD44v8-10, binds to and stabilizes the cystine transporter subunit (xCT), producing reduced glutathione and thereby enhancing the antioxidant defense of cancer stem cells. Pharmacological inhibition of xCT by sulfasalazine suppresses tumor growth, survival, and resistance to chemotherapy. We investigated whether the CD44v-xCT axis contributes to PAH pathogenesis. CD44v was predominantly expressed on endothelial-to-mesenchymal transition (EndMT)-like cells in the neointimal layer of PAH affected pulmonary arterioles. In vitro, CD44 standard form and CD44v were induced as a result of EndMT. Among human pulmonary artery endothelial cells that have undergone EndMT, CD44v+ cells showed high levels of xCT expression on their cell surfaces and high concentrations of glutathione for survival. This made CD44v+ cells the most vulnerable target for sulfasalazine. CD44v+xCThi cells showed the highest expression levels of proinflammatory cytokines, antioxidant enzymes, antiapoptotic molecules, and cyclin-dependent kinase inhibitors. In the Sugen5416/hypoxia mouse model, CD44v+ cells were present in the thickened pulmonary vascular wall. The administration of sulfasalazine started either at the same time as "Sugen5416" administration (a prevention model) or after the development of pulmonary hypertension (a reversal model) attenuated the muscularization of the pulmonary vessels, decreased the expression of markers of inflammation, and reduced the right ventricular systolic pressure, while reducing CD44v+ cells. In conclusion, CD44v+xCThi cells appear during EndMT and in pulmonary hypertension tissues. Sulfasalazine is expected to be a novel therapeutic agent for PAH, most likely targeting EndMT-derived CD44v+xCThi cells.

Abstract 1165: Differential expression of CD44 variants drive the progression, invasion, drug-resistance and stemness characteristics in human oral squamous cell carcinoma

Abstract Purpose: Cluster of differentiation 44 (CD44) is a cell-surface glycoprotein and plays role in the progression and severity of oral squamous cell carcinoma (OSCC). Here we report differential function of CD44 variants in OSCC progression. Materials and methods: The expression of CD44 standard (CD44s) and variants (CD44v4, CD44v6); the activation of signaling pathways (MAPK, PI3K); the cell viability; and the MMP-9/-2 activity were assessed using RT-PCR, immunohistochemistry, Western blotting, MTT assay and gelatin zymography. These experiments were carried out using fresh human OSCC tissue specimens, including adjacent normal, noninvasive (N0), invasive tumor samples (N1-3) and chemo-radiation resistant tumor samples (RCRT). We also used OSCC cell lines, including parental (SCC9/SCC4) and Cisplatin-resistant (CisR-SCC9/-SCC4) to confirm our observation with human tissues. Knock down of CD44 variants or inactivation of MAPK/PI3K pathways was achieved for in vitro analysis in OSCC cells. Summary: Differential expressions of CD44 variants (v4and v6) were associated with overall oral cancer aggressiveness. CD44v4 expression was positively correlated with the activation of MAPK pathway causing chemoresistance. Conversely, CD44v6 expression and activation of PI3K-Akt caused invasiveness of OSCC. Finally, an overlapping role of two major signal transduction pathways such as MAPK and PI3K, that impinge on the variants/ isoforms expressions of a single gene i.e. CD44 that leads to the OSCC aggressiveness. Conclusion: Collectively, these results established that CD44 variants (v4and v6) expression driven by MAPK/PI3K are associated with the overall progression, drug-resistance, invasion, and stemness characteristics leading to the aggressiveness of OSCC. Hence targeting these pathways may be exploited for treating OSCC. Keywords: OSCC, CD44s, CD44v4/6, MAPK, PI3K, Notch, Chemoresistance, Invasion/migration, stemness, aggressiveness of OSCC. Note: This abstract was not presented at the meeting. Citation Format: Tanushree Kashyap, Siddavaram Nagini, Ajay Rana, Rajakishore Mishra. Differential expression of CD44 variants drive the progression, invasion, drug-resistance and stemness characteristics in human oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1165.

Deficiency of Stomach-Type Claudin-18 in Mice Induces Gastric Tumor Formation Independent of Hpylori Infection.

Background & AimsEpithelial cells are joined by tight junctions (TJs) to form a cell sheet. In the stomach, epithelial cell sheet forms an essential barrier against gastric material, including gastric acid. Although the decreased expression of stomach-type claudin-18 (stCldn18), a TJ protein, is generally observed in human gastritis and gastric cancer, its pathological roles are not fully understood. We previously reported that mice lacking stCldn18 (stCldn18-/-) exhibit gastric acid leakage through TJs, which induces active gastritis at a young age. Here, we examined the gastric pathologies in mice after long-term stCldn18 deficiency.MethodsThe gastric pathologies in stCldn18-/- mice were sequentially analyzed from youth to old age, and compared to those in humans. To examine the relationship between stCldn18 deficiency-induced gastric pathologies and Wnt-dependent tumorigenesis, we generated Wnt1-overexpressing stCldn18-/- mice.ResultsStCldn18-/- mice developed chronic active gastritis at middle age, with expression of the chemoattractant CCL28. At old age, 20-30% of these mice developed gastric tumors with CXCL5 expression, indicative of EMT. In this process, spasmolytic polypeptide-expressing metaplasia (SPEM) cells appeared. Increased expressions of CD44-variants, TLR2, and CXCL5 indicated age-dependent changes in cell characteristics. Some features of the stCldn18-/- mouse gastric tumorigenesis resembled H pylori-infection-related human carcinogenesis. The gastric tumorigenesis was accelerated in Wnt1-overexpressing stCldn18-/- mice, indicating that Wnt is involved in the stCldn18-/- mouse gastric tumorigenesis.ConclusionsStCldn18 deficiency induced gastric tumorigenesis in mice without H pylori infection. Our findings revealed that several signaling networks, including the cytokine-, stemness-, and Wnt-signaling pathways, may be activated under the stCldn18-deficiency-induced chronic active gastritis to accelerate the gastric tumorigenesis.

Crosstalk between Raf-MEK-ERK and PI3K-Akt-GSK3β signaling networks promotes chemoresistance, invasion/migration and stemness via expression of CD44 variants (v4 and v6) in oral cancer

BackgroundThe cell-surface glycoprotein CD44 is an important oral cancer stem cell (OCSC) marker and plays significant role in oral squamous cell carcinoma (OSCC) aggressiveness, however, the regulation of CD44 is incompletely understood. MethodsIn the present study, 145 fresh human OSCC tissue specimens, including 18 adjacent normal, 42 noninvasive (N0), 53 invasive tumor samples (N1-3) and 32 chemo-radiation resistant samples (RCRT), were included. The expression of CD44 standard (CD44s) and variants (CD44v4, CD44v6); the activation of pERK1/2, GSK3β, NICD (Notch) pathways; the cell viability; and the MMP-9/-2 activity were assessed using RT-PCR, immunohistochemistry, Western blotting, MTT assay and gelatin zymography. OSCC cell lines, including parental (SCC9/SCC4) and Cisplatin-resistant (CisR-SCC9/-SCC4) cells, were used. Knock down of CD44v4/CD44v6 (by siRNA) or inactivation of MAPK/PI3K pathways using specific PD98059/LY294002 was achieved for in vitro analysis of chemoresistance and invasion/migration. ResultsElevated CD44 variants were associated with overall OSCC progression, chemoresistance and invasion. Positive correlations were observed, mainly between the expression of CD44v4 and the activation of ERK1/2 causing chemoresistance, whereas CD44v6 expression and inactivation of GSK3β caused invasiveness of OSCC. Cisplatin resistant, CisR-SCC9/SCC4 cell lines showed OCSC properties. Inhibition of MEK/ERK1/2 by SMI or knock down (KD) of CD44v4 by siRNA reversed cisplatin-resistance, whereas blocking the PI3K/Akt/GSK3β pathway by SMI or KD of CD44v6 isoforms by respective siRNA diminished invasion/metastasis potential. ConclusionCollectively, our results demonstrated that CD44v4 expression is more linked with ERK1/2 activation and promote cisplatin resistance, whereas CD44v6 expression is associated primarily with PI3K/Akt/GSK3β activation and driving tumor invasion/migration.