Co-expression of CD44v6 and MMP2 predicts lung metastasis and unfavorable prognosis in osteosarcoma.

Aim: To evaluate the prognostic significance of CD44 variant v6 (CD44v6) and matrix metalloproteinases 2 (MMP2) expression in patients with surgically resected osteosarcoma. Methods: CD44v6 and MMP2 expression were immunohistochemically detected in 113 primary osteosarcoma patients at our institute between 2001 and 2019. Results: Both CD44v6 and MMP2 were independent predictors for metastasis-free and overall survival. An extended predictive range and improved sensitivity were observed when the combined effects of CD44v6 and MMP2 were considered. Specifically, patients with CD44v6+ and MMP2+ expression were more susceptible to lung metastasis and exhibited the poorest survival rates compared with the other groups. Conclusion: The combination of CD44v6 and MMP2 may serve as a precise prognostic indicator for predicting metastatic progression and survival outcomes in patients with osteosarcoma.

The most common type of bone cancer in children, teens and young adults is osteosarcoma, which often spreads to the lungs. With proper chemotherapy and surgery, many patients can recover, but if the diagnosis and treatment process go wrong, it could have serious consequences. The most common symptoms of osteosarcoma in its early stages are pain and swelling. The pain usually comes and goes, which can be easily mistaken for growing pains, resulting in a delayed diagnosis. In patients with metastatic (cancer cells spreading from the primary site to other parts of the body) osteosarcoma, the number of metastatic sites and whether they can be completely removed through surgery are factors that affect prognosis. So, starting appropriate treatment early for patients could effectively reduce tumor spread and increase survival time.

Sulforaphane inhibits CD44v6/YAP1/TEAD signaling to suppress the cancer phenotype.

Sulforaphane (SFN) is a promising cancer prevention and treatment agent that strongly suppresses the cutaneous squamous cell carcinoma (CSCC) cell cancer phenotype. We previously showed that yes-associated protein 1 (YAP1)/TEAD signaling is a key procancer stimulator of the aggressive CSCC cell cancer phenotype. However, SFN-responsive upstream regulators of YAP1/TEAD signaling are not well characterized and so there is a pressing need to identify these factors. We show that CD44v6 knockdown reduces YAP1/TEAD-dependent transcription and target gene expression, and that this is associated with reduced spheroid formation, invasion and migration. CD44v6 knockout cell lines also display reduced YAP1/TEAD activity and target gene expression and attenuated spheroid formation, invasion, migration and tumor formation. An important finding is that SFN treatment suppresses CD44v6 level leading to a reduction in YAP1/TEAD signaling and marker gene expression. Sox2 level and epithelial-mesenchymal transition (EMT) are also reduced. Forced expression of constitutive active YAP1 in CD44v6 knockdown cells partially restores the aggressive cancer phenotype. These important findings suggest that CD44v6 drives YAP1/TEAD signaling to enhance the CSCC cell cancer phenotype and that SFN treatment reduces CD44v6 level/function which, in turn, reduces YAP1/TEAD signaling leading to reduced stemness, EMT and tumor growth.

The clinical significance of CD44v6 in malignant and benign primary bone tumors.

BACKGROUND: The objective of this study was to assess the expression profile of CD44v6, a potential cancer stem cell marker, and its diagnostic and predictive significance in three distinct types of primary bone tumors. METHODS: In this study, we utilized real-time qRT-PCR and immunohistochemistry to examine the gene and protein levels of CD44v6 in a total of 138 fresh bone tissues. This included 69 tumor tissues comprising osteosarcoma (N = 23), chondrosarcoma (N = 23), and GCT (N = 23), as well as 69 corresponding non-cancerous tumor margins. Furthermore, we investigated the circulating level of CD44v6 by isolating peripheral blood mononuclear cells from 92 blood samples. Among these, 69 samples were obtained from patients diagnosed with primary bone tumors, while the remaining 23 samples were from healthy donors. The primary objectives of our investigation were to assess the correlation between CD44v6 expression levels and clinic-pathological features of the patients, as well as to evaluate the diagnostic and predictive values of CD44v6 in this context. RESULTS: In patients with osteosarcoma and chondrosarcoma tumors, both the gene and protein expression of CD44v6 were found to be significantly higher compared to the GCT group. Furthermore, the circulating level of CD44v6 was notably elevated in patients diagnosed with osteosarcoma and chondrosarcoma in comparison to the GCT group and patients with malignant tumor characteristics. Additionally, we observed a strong correlation between the gene and protein levels of CD44v6 and important tumor indicators such as tumor grade, metastasis, recurrence, and size at the tumor site. CD44v6 shows potential in differentiating patients with bone tumors from both control groups and tumor groups with severe and invasive characteristics from those with non-severe features. Importantly, the expression level of CD44v6 also demonstrated predictive value for determining tumor grade and the likelihood of recurrence. CONCLUSION: CD44v6 is likely to play a role in the development of primary bone tumors and has the potential to serve as a diagnostic biomarker for bone cancer. However, to obtain more accurate and conclusive findings, further mechanistic investigations involving larger population samples are necessary.

Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C44Mab-9 for Multiple Applications against Colorectal Carcinomas.

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (KD) of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10-9 M, 1.7 × 10-8 M, and 2.3 × 10-8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.

Next Generation CD44v6-Specific CAR-NK Cells Effective against Triple Negative Breast Cancer.

There is a medical need to develop new and effective therapies against triple-negative breast cancer (TNBC). Chimeric antigen receptor (CAR) natural killer (NK) cells are a promising alternative to CAR-T cell therapy for cancer. A search for a suitable target in TNBC identified CD44v6, an adhesion molecule expressed in lymphomas, leukemias and solid tumors that is implicated in tumorigenesis and metastases. We have developed a next-generation CAR targeting CD44v6 that incorporates IL-15 superagonist and checkpoint inhibitor molecules. We could show that CD44v6 CAR-NK cells demonstrated effective cytotoxicity against TNBC in 3D spheroid models. The IL-15 superagonist was specifically released upon recognition of CD44v6 on TNBC and contributed to the cytotoxic attack. PD1 ligands are upregulated in TNBC and contribute to the immunosuppressive tumor microenvironment (TME). Competitive inhibition of PD1 neutralized inhibition by PD1 ligands expressed on TNBC. In total, CD44v6 CAR-NK cells are resistant to TME immunosuppression and offer a new therapeutic option for the treatment of BC, including TNBC.

Downregulation of Hyaluronic acid-CD44 signaling pathway in cervical cancer cell by natural polyphenols Plumbagin, Pongapin and Karanjin.

Hyaluronic acid (HA)-CD44 pathway showed association with several malignancies. The natural polyphenols Plumbagin, Pongapin and Karanjin showed anti-cancer activities in different tumors including cervical carcinoma. To understand their mechanism of anti-cancer activity, the effect of the compounds on HA-CD44 pathway was analyzed in cervical cancer cell line HeLa. The mRNA expression of three different isoforms of CD44 i.e., CD44s, CD44v3, and CD44v6, was differentially downregulated by the compounds. This was validated by Western blot and immunocytochemical analysis of CD44s.The low molecular weight HA (LMW-HA) showed growth promoting activity in HeLa at low concentration, whereas high molecular weight HA (HMW-HA) had no such effect. The compounds could preferentially downregulate the LMW-HA level in HeLa, as evident in the cell as well as in the cell-free conditioned medium. Concentration-dependent upregulation of HA synthase-2 (HAS2) was seen in the cell by the compounds, whereas differential downregulation of hyalurinidases 1-4 (HYAL 1-4), predominantly HYAL1, were seen. The compounds could also downregulate the downstream target of the pathway p-AKT (T-308) in concentration-dependent manner. Thus, the compounds could attenuate the HA-CD44 pathway in HeLa cell to restrict the tumor growth.

CD44 variant 6 is associated with prostate cancer growth and chemo-/radiotherapy response in vivo.

We have previously demonstrated that CD44 variant 6 (CD44v6) is associated with prostate cancer (CaP) growth and therapeutic resistance in vitro, however, the role of CD44v6 in CaP in vivo is not fully understood. The purpose of this study is to investigate the effect of CD44v6 on CaP growth and chemo-/radiotherapy response in NOD/SCID mouse models in vivo and to validate its role as a therapeutic target for CaP therapy. CD44v6 was knocked down in PC-3M CaP cell line using short hairpin RNA. Subcutaneous (s.c.) and orthotopic CaP mouse xenografts were established. The effect of CD44v6 knockdown (KD) on tumour growth was evaluated in both s.c. and orthotopic models. Chemo-/radiotherapy response was evaluated in the s.c. model. Association of CD44v6 with PI3K/Akt pathway was validated using immunohistochemistry staining. We found that KD of CD44v6 significantly reduced tumour growth in both models, and enhanced the sensitivity of tumours to chemotherapy and radiotherapy in the s.c. model. In addition, we demonstrated that KD of CD44v6 is associated with downregulation of the PI3K/Akt/mTOR pathway. Our data confirm that CaP growth and chemo-/radiosensitivity in vivo is associated with CD44v6, which holds great promises as a therapeutic target in the treatment of CaP.

FOLFOX Therapy Induces Feedback Upregulation of CD44v6 through YB-1 to Maintain Stemness in Colon Initiating Cells.

Cancer initiating cells (CICs) drive tumor formation and drug-resistance, but how they develop drug-resistance characteristics is not well understood. In this study, we demonstrate that chemotherapeutic agent FOLFOX, commonly used for drug-resistant/metastatic colorectal cancer (CRC) treatment, induces overexpression of CD44v6, MDR1, and oncogenic transcription/translation factor Y-box-binding protein-1 (YB-1). Our study revealed that CD44v6, a receptor for hyaluronan, increased the YB-1 expression through PGE2/EP1-mTOR pathway. Deleting CD44v6, and YB-1 by the CRISPR/Cas9 system attenuates the in vitro and in vivo tumor growth of CICs from FOLFOX resistant cells. The results of DNA:CD44v6 immunoprecipitated complexes by ChIP (chromatin-immunoprecipitation) assay showed that CD44v6 maintained the stemness traits by promoting several antiapoptotic and stemness genes, including cyclin-D1, BCL2, FZD1, GINS-1, and MMP9. Further, computer-based analysis of the clones obtained from the DNA:CD44v6 complex revealed the presence of various consensus binding sites for core stemness-associated transcription factors "CTOS" (c-Myc, TWIST1, OCT4, and SOX2). Simultaneous expressions of CD44v6 and CTOS in CD44v6 knockout CICs reverted differentiated CD44v6-knockout CICs into CICs. Finally, this study for the first time describes a positive feedback loop that couples YB-1 induction and CD44 alternative splicing to sustain the MDR1 and CD44v6 expressions, and CD44v6 is required for the reversion of differentiated tumor cells into CICs.

Elucidation of the mechanism underlying CD44v6-induced transformation of IEC-6 normal intestinal epithelial cells.

The transformation abilities of CD44s and CD44v6 in normal intestinal epithelial cells have not yet been reported. Herein, we established both CD44s and CD44v6 overexpressing stable clones from rat IEC-6 cells and demonstrated that the CD44v6 clones had higher saturation density and anchorage independence. Additionally, CD44v6 clones were more resistant to oxaliplatin and irinotecan which might be attributed to a significantly increased B-cell lymphoma 2 level and a reduced DNA damage response in these cells. Moreover, c-Met and vascular endothelial growth factor receptor 2 signalings were involved in modulating the saturation density in CD44v6 clones. Interestingly, higher activation of both AKT and extracellular-signal-regulated kinase (ERK) were detected in CD44v6 clones which might account in part for the cell density-independent nuclear localization of Yes-associated protein (YAP). To no surprise, increases of both saturation density and anchorage independence in CD44v6 clones were markedly diminished by PI3K, AKT, MEK, and ERK inhibitors as well as YAP knockdown. By contrast, overexpression of a constitutively active YAP robustly increased the aforementioned phenotypes in IEC-6 cells. Collectively, our results suggest that upregulation of CD44v6, but not CD44s, induces the transformation of normal intestinal epithelial cells possibly via activating the c-Met/AKT/YAP pathway which might also explain the important role of CD44v6 in the initiation of various carcinomas.

Structural Characterization of the CD44 Stem Region for Standard and Cancer-Associated Isoforms.

CD44 is widely expressed in most vertebrate cells, whereas the expression of CD44v6 is restricted to only a few tissues and has been considered to be associated with tumor progression and metastasis. Thus, CD44v6 has been recognized as a promising prognostic biomarker and therapeutic target for various cancers for more than a decade. However, despite many experimental studies, the structural dynamics and differences between CD44s and CD44v6, particularly in their stem region, still remain elusive. Here, a computational study was conducted to address these problems. We found that the stem of CD44s adopted predominantly two conformations, one featuring antiparallel ß-sheets and the other featuring parallel ß-sheets, whereas the stem of CD44v6 adopted mainly one conformation with relatively highly suppressed ß-sheet contents. Moreover, Phe215 was found to be essential in the ß-sheets of both CD44s and CD44v6. We finally found intramolecular Phe215-Trp224 hydrogen-bonding interactions and hydrophobic interactions with Phe215 that cooperatively drove conformational differences upon the addition of the v6 region to CD44. Our study elucidated the structural differences between the stem regions of CD44s and CD44v6 and thus can offer useful structural information for drug design to specifically target CD44v6 in promising clinical applications.

Expression of CD44v6 and lymphatic vessel density in early gastric cancer tissues and their clinical significance.

OBJECTIVE: To explore the relationships between expression of CD44v6, lymphatic vessel density (LVD) and the clinicopathological parameters of patients. METHODS: One hundred early gastric cancer tissues, 55 high-grade gastric intraepithelial neoplasia (HGIN) tissues, 60 low-grade gastric intraepithelial neoplasia (LGIN) tissues and 60 chronic superficial gastritis tissues were collected and set as gastric cancer group, HGIN group, LGIN group and gastritis group respectively. The expression of CD44v6 and LVD of patients in all the groups were detected using two-step immunohistochemical method to analyze the relationships between the expression of CD44v6 and lymphatic vessel density in early gastric cancer tissues and their relationships with the clinicopathological parameters of patients. The values of LVD in predicting lymph node metastasis in early gastric cancer were evaluated using receiver operating characteristic (ROC) curve. RESULTS: The positive expression of CD44v6 and LVD in the gastritis group, LGIN group, HGIN group and gastric cancer group gradually increased. The positive expression of CD44v6 and LVD in early gastric cancer tissues were in no correlation with the gender, age, tumor site, maximum diameter, differentiation degree and invasion depth (P>0.05) and in a correlation with lymphatic metastasis and lymphatic vessel invasion (P<0.06). The positive expression of CD44v6 in the early gastric cancer tissues was in a positive correlation with LVD (P<0.05). The analysis of ROC curves suggested that the area under ROC curve of predicting lymphatic metastasis of early gastric cancer with LVD was 0.837 (95% CI: 0.756~0.910), and the cut-off value was 14; the corresponding sensitivity and specificity were 63.6% and 90.2 respectively. CONCLUSION: The expression of CD44v6 and LVD in early gastric cancer tissues are in a close correlation with the clinicopathologic features, and joint detection of expression of CD44v6 and LVD can be taken as the indicator of gastric cancer metastasis.

Novel role of O-glycosyltransferases GALNT3 and B3GNT3 in the self-renewal of pancreatic cancer stem cells.

BACKGROUND: Glycosylation plays a critical role in the aggressiveness of pancreatic cancer (PC). Emerging evidences indicate significant involvement of cancer stem cells (CSCs) in PC aggressiveness. However, the importance of glycosylation in pancreatic cancer stem cells (PCSCs) is yet to be addressed. Hence, we evaluated the potential role of glycosylation in maintenance of stemness of PCSCs. METHODS: Effect of glycosylation specific inhibitors on growth and PCSCs of PC cells was assessed by MTT assay and Side Population (SP) analysis. Isolated PCSCs/SP were characterized using molecular and functional assays. Expression of tumor-associated carbohydrate antigens (TACAs) was analyzed in PCSCs by western blotting. Effect of tunicamycin on PCSCs was analyzed by tumorsphere, clonogenicity, migration assay and immunoblotting for CSCs markers. The differential expression of glycogenes in PCSCs compared to non-CSCs were determined by RT-qPCR, immunoblotting and immunofluorescence. Co-expression of GALNT3 and B3GNT3 with CD44v6 was assessed in progression stages of KrasG12D; Pdx-1-Cre (KC) and KrasG12D; p53R172H; Pdx-1-Cre (KPC) tumors by immunofluorescence. Transient and CRISPR/Cas9 silencing of GALNT3 and B3GNT3 was performed to examine their effect on CSCs maintenance. RESULTS: Inhibition of glycosylation decreased growth and CSCs/SP in PC cells. PCSCs overexpressed CSC markers (CD44v6, ESA, SOX2, SOX9 and ABCG2), exhibited global expressional variation of TACAs and showed higher self-renewal potential. Specifically, N-glycosylation inhibition, significantly decreased tumorsphere formation, migration, and clonogenicity of PCSCs, as well as hypo-glycosylated CD44v6 and ESA. Of note, glycosyltransferases (GFs), GALNT3 and B3GNT3, were significantly overexpressed in PCSCs and co-expressed with CD44v6 at advanced PDAC stages in KC and KPC tumors. Further, GALNT3 and B3GNT3 knockdown led to a decrease in the expression of cell surface markers (CD44v6 and ESA) and self-renewal markers (SOX2 and OCT3/4) in PCSCs. Interestingly, CD44v6 was modified with sialyl Lewis a in PCSCs. Finally, CRISPR/Cas9-mediated GALNT3 KO significantly decreased self-renewal, clonogenicity, and migratory capacity in PCSCs. CONCLUSIONS: Taken together, for the first time, our study showed the importance of glycosylation in mediating growth, stemness, and maintenance of PCSCs. These results indicate that elevated GALNT3 and B3GNT3 expression in PCSCs regulate stemness through modulating CSC markers.

Prognostic significance of elevated serum CD44 levels in patients with oral squamous cell carcinoma.

BACKGROUND: Availability of reliable methods distinguishing high-risk recurrent tumours from regressive tumours prior to surgery could help in better management of the disease. This study was aimed to estimate pre-surgical serum CD44 concentration and assess the possibility of using it as a non-invasive prognostic tool in oral cancer. METHODS: ELISA was performed on pre-surgical serum samples from 64 primary oral cancer patients and 16 healthy individuals to estimate soluble CD44 levels. Immunohistochemistry was performed on parallel 64 solid tumours and 10 recurrent tumours. All patients clinically followed up for median period of 19.2 months and obtained prognostic information correlated with CD44 concentration in serum as well as in tumours. RESULTS: Serum CD44 concentration was found significantly high in patients as compared to healthy individuals (P < .001) and also in patients whose disease locally recurred as compared to those did not recur (P = 0026). High serum CD44 concentration inversely affected on patients survival (P = .032). CD44v6 staining intensity was detected significantly high in recurrent tumours as compared to primary tumours (P < .001), and it also correlated with poor survival (P < .001). Furthermore, in combination, patients with increased CD44 concentration in serum and CD44v6 expression in tumours significantly correlated with local recurrence (P < .001) and poor survival (P < .001). CONCLUSION: Our data suggest that the ELISA-based estimation of pre-surgical serum CD44 concentration could be a non-invasive reliable method distinguishing high-risk recurrent tumours which can further assist in post-surgery treatment planning.

Spatial distribution of osteopontin, CD44v6 and podoplanin in the lining epithelium of odontogenic keratocyst, and their biological relevance.

BACKGROUND AND AIMS: The odontogenic keratocyst (OKC) remains the most challenging jaw cyst to treat because of its locally-aggressive behaviour and high recurrence potential. Emerging evidence suggests that osteopontin, its receptors CD44v6 and integrin αv, and podoplanin, have a role in the local invasiveness of this cyst. However the spatial distribution characteristics of these pro-invasive markers in the lining epithelium of OKC, and their association with the clinicopathologic parameters of OKC are largely unexplored. This study sought to address these issues in comparison with dentigerous cysts (DCs) and radicular cysts (RCs) and to evaluate their biological relevance. METHODS: A sample consisting of 20 OKC cases, 10 DCs and 10 RCs was subjected to immunohistochemical staining for osteopontin, CD44v6 and integrin αv, and podoplanin, and semiquantitative analysis was performed. RESULTS: All factors (except integrin αv) were detected heterogeneously in the constitutive layers of the lining epithelium in all three cyst types. Key observations were significant upregulation of CD44v6 and podoplanin in OKC compared to DCs and RCs, suggesting that these protein molecules may play crucial roles in promoting local invasiveness in OKC (P<0.05). Osteopontin underexpression and distribution patterns were indistinctive among all three cysts indicating its limited role as pro-invasive factor. Clinical parameters showed no significant correlations with all protein factors investigated. CONCLUSIONS: Present findings suggest that an osteopontinlow CD44v6high and podoplaninhigh immunoprofile most probably represent epithelial signatures of OKC and are markers of local invasiveness in this cyst.

Expression of CD44 variant 6 and its prognostic value in benign and malignant endometrial tissue.

PURPOSE: CD44 expression in both the early and metastatic phases of many epithelial and non-epithelial cancers is strongly prognostic. The objective of the study is to evaluate whether there is any relationship between the expression of CDD44v6 and endometrial cancer (EC) staging and prognosis. METHODS: This retrospective study included 60 EC patients for whom surgical staging was performed between 2000 and 2006. Twenty-eight randomly selected patients with normal endometria served as the control group. We immunohistochemically evaluated membranous and cytoplasmic CD44v6 staining in tissue paraffin blocks. The results were graded as positive or negative. RESULTS: Membranous staining in both advanced and early stage EC patients was significantly higher than that in the control group (p = 0.002). The extent of either membranous or cytoplasmic staining in both advanced- and early stage patients did not differ significantly by age, tumor grade, stage, extent of myometrial invasion, lymph node involvement, cytology, adnexal involvement, or omental spreading. In advanced-stage patients, neither papillary serous not clear cell cancers exhibited cytoplasmic staining. CONCLUSIONS: CD44v6 membranous staining can be useful for differentiating malignant from benign endometrial tissue. However, staining is not associated with EC staging or prognosis.

Upregulation of CD44v6 contributes to acquired chemoresistance via the modulation of autophagy in colon cancer SW480 cells.

The CD44 isoform containing variant exon v6 (CD44v6) plays an important role in the progression, metastasis, and prognosis of colorectal cancer (CRC). Recently, it was found that CD44v6 is involved in acquired drug resistance. This study aimed to investigate the molecular mechanism of CD44v6 in the resistance of CRC cells to chemotherapy. A stable CD44v6 overexpression model in SW480 cells was established via lentiviral transduction. The chemosensitivity of cells to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP) was determined by cell counting kit (CCK)-8, lactate dehydrogenase (LDH) release, and colony formation assays. Immunohistochemical staining of CD44v6 was performed in human CRC tissues. The key components in cell apoptosis, drug efflux and metabolism, mismatch repair, autophagy, epithelial-mesenchymal transition (EMT), and the PI3K-Akt and MAPK-Ras-Erk1/2 pathways were assessed using flow cytometry, quantitative real-time polymerase chain reaction (PCR), and western blot assays. The CD44v6 overexpression cells showed a higher viability, a lower LDH release rate, and an increased clonogenicity than the control cells under drug treatment. Moreover, overexpression of CD44v6 resulted in enhanced autophagy flux, EMT, and phosphorylation of Akt and Erk in the presence of drugs. Furthermore, high CD44v6 expression in the primary tumor was closely associated with an early recurrence in CRC patients who underwent curative surgery and adjuvant chemotherapy. In conclusion, overexpression of CD44v6 contributes to chemoresistance in SW480 cells under cytotoxic stress via the modulation of autophagy, EMT, and activation of the PI3K-Akt and MAPK-Ras-Erk pathways.

Molecular imaging of EGFR and CD44v6 for prediction and response monitoring of HSP90 inhibition in an in vivo squamous cell carcinoma model.

PURPOSE: Heat shock protein 90 (HSP90) is essential for the activation and stabilization of numerous oncogenic client proteins. AT13387 is a novel HSP90 inhibitor promoting degradation of oncogenic proteins upon binding, and may also act as a radiosensitizer. For optimal treatment there is, however, the need for identification of biomarkers for patient stratification and therapeutic response monitoring, and to find suitable targets for combination treatments. The aim of this study was to assess the response of surface antigens commonly expressed in squamous cell carcinoma to AT13387 treatment, and to find suitable biomarkers for molecular imaging and radioimmunotherapy in combination with HSP90 inhibition. METHODS: Cancer cell proliferation and radioimmunoassays were used to evaluate the effect of AT13387 on target antigen expression in vitro. Inhibitor effects were then assessed in vivo in mice-xenografts. Animals were treated with AT13387 (5 × 50 mg/kg), and were imaged with PET using either (18)F-FDG or (124)I-labelled tracers for EGFR and CD44v6, and this was followed by ex-vivo biodistribution analysis and immunohistochemical staining. RESULTS: AT13387 exposure resulted in high cytotoxicity and possible radiosensitization with IC50 values below 4 nM. Both in vitro and in vivo AT13387 effectively downregulated HSP90 client proteins. PET imaging with (124)I-cetuximab showed a significant decrease of EGFR in AT13387-treated animals compared with untreated animals. In contrast, the squamous cell carcinoma-associated biomarker CD44v6, visualized with (124)I-AbD19384 as well as (18)F-FDG uptake, were not significantly altered by AT13387 treatment. CONCLUSION: We conclude that AT13387 downregulates HSP90 client proteins, and that molecular imaging of these proteins may be a suitable approach for assessing treatment response. Furthermore, radioimmunotherapy targeting CD44v6 in combination with AT13387 may potentiate the radioimmunotherapy outcome due to radiosensitizing effects of the drug, and could potentially lead to a lower dose to normal tissues.

E-Cadherin, CD44v6, and Insulin-Like Growth Factor-II mRNA-Binding Protein 3 Expressions in Different Stages of Hydatidiform Moles.

E-cadherin, CD44v6, and IMP3 expression in partial, complete, and invasive hydatidiform moles (HMs) was evaluated. High E-cadherin expression with low CD44v6 expression was observed in partial, complete, and invasive HMs, as well as in normal placental tissues; and there was no significant difference in E-cadherin and CD44v6 expression among the four groups. However, IMP3 expression was gradually decreased in the order of normal placental tissues, partial HMs, complete HMs, and invasive HMs; wherein, invasive HMs had the lowest level. Low IMP3 expression may serve as a prognostic biomarker for HMs, and IMP3 may play a certain role in HMs progression.

Prognostic significance of CD44v6, p63, podoplanin and MMP-9 in oral squamous cell carcinomas.

OBJECTIVES: To analyse the expression of the CD44v6, p63, podoplanin and MMP-9, and their prognostic significance in patients with oral squamous cell carcinomas (OSCC). MATERIAL AND METHODS: Immunohistochemistry technique was performed on 60 OSCC for detection of CD44v6, p63, podoplanin and MMP-9 proteins. Extent and intensity of staining were evaluated in tumour cells and were compared with patients' clinical-pathological characteristics and survival. RESULTS: CD44v6 expression was detected at the membrane of tumour cells of 94% cases. Nuclear expression of p63 protein was present in 96.5%. Podoplanin was observed at the membrane of tumour cells of 94% cases. MMP-9 was found in the cytoplasm of tumour cells in 83.7% cases. A high level of expression (67%-89%) in all four proteins was noted. Podoplanin was associated with the expression of MMP-9 (P = 0.010) and both were associated with lymph node metastasis (P = 0.011 and P = 0.018, respectively). Co-expression of podoplanin/MMP-9 was an adverse independent prognostic factor for cancer-specific survival (P = 0.008) and recurrence-free survival (P = 0.042). CONCLUSION: Podoplanin and MMP-9 together could contribute to tumour progression and dissemination of OSCC. Their combined overexpression showed an adverse effect on survival, suggesting that they could be regarded as important prognostic biomarkers in OSCC.

The expression of CD44v6 in colon: from normal to malignant.

CD44v6, an integral transmembrane protein belonging to a family of adhesion molecule receptors, plays an important role in tumor growth, progression and metastasis. The purpose of this study was to evaluate the expression of CD44v6 in normal, hyperplastic, adenomatous, and malignant colonic epithelium and to determine its correlation with tumor pathologic stage and lymph node metastasis. We examined the immunohistochemical expression of CD44v6 in normal colonic tissue (n = 25), hyperplastic polyps (n = 45), tubular adenomas (n = 57), tubulovillous adenomas (n = 25), villous adenomas (n = 9), adenocarcinomas stage I (n = 26), adenocarcinomas stage III (n = 26), and lymph node metastasis (n = 26). The percentage of positive cells and the staining intensity were assessed and scored. Statistical analysis was performed using logistic regression and McNemar test. All normal colonic tissue and hyperplastic polyps showed CD44v6 staining confined to the base of the crypt. In tubular adenomas, the dysplastic surface adenomatous epithelium expressed CD44v6 in 49 (86%) cases. CD44v6 was expressed in the glandular areas of tubulovillous adenomas in 21 (84%) cases and in the villous portion in 18 (72%) cases. All villous adenomas expressed CD44v6. CD44v6 was expressed in 23 (88%) cases of stage I adenocarcinomas, in 24 (92%) cases of stage III adenocarcinomas, and in 9 (35%) cases of metastatic adenocarcinomas. We concluded that the gain of CD44v6 expression in premalignant and malignant colonic lesions suggests that CD44v6 may be functionally involved in the adenoma-to-carcinoma progression. CD44v6 did not correlate to tumor pathologic stage and is lost during the acquisition of migratory function by metastatic tumor cells.