Selective killing of breast cancer cells expressing activated CD44 using CD44 ligand-coated nanoparticles in vitro and in vivo

The cell surface glycoprotein CD44 is expressed in cancer cells and has been used as a therapeutic target in preclinical studies. However, the ubiquitous expression of CD44 in numerous cell types, including hematopoietic cells, has hindered its application in targeted therapy. Here, we demonstrated that CD44 was activated on breast cancer cells but was inactive on normal cells in vitro and in vivo. We analyzed 34 clinical primary tumor and normal breast tissues and demonstrated that CD44 was in an active state on breast cancer cells but in an inactive state on normal cells. Furthermore, based on the binding property of CD44 with its ligand hyaluronan (HA), we self-assembled HA-coated nanoparticles and studied their selective targeting efficacy. Our results indicate that HA-coated nanoparticles bearing the CD44 ligand selectively targeted cancer cells both in vitro and in vivo, killing breast cancer cells while sparing normal cells. Our study suggested that the active state of CD44 plays a crucial role in the selective targeting of breast cancer cells by avoiding nonspecific toxicity to CD44-quiescent normal cells. These findings may provide a new idea for the selective targeting of cancer cells in other human cancers.     

CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

CD44 is a cell surface antigen expressed on acute myeloid leukemia cells and is used as a marker to isolate leukemia stem cells. CD44 ligation with the antibody A3D8 has been found to induce apoptosis in human acute promyelocytic leukemia (APL) cells via activation of caspase-8. The mechanism of A3D8-induced caspase-8 activation was studied in APL NB4 cells. A3D8 induces lipid raft clustering which causes Fas aggregation as determined with a confocal microscope. A3D8-induced apoptosis is abrogated by the lipid raft disrupting agent methyl-β-cyclodextrin and the caspase-8 inhibitor Z-IETD-fmk. Western blot analysis reveals that A3D8 binds to the standard form of CD44 (CD44s). HL-60 cells without detectable CD44s protein are not responsive to A3D8-induced apoptosis. SKNO-1 cells containing higher level of CD44s protein are more sensitive to A3D8-induced apoptosis than NB4 cells. These results indicate that A3D8 induces apoptosis in leukemia cells through caspase-8 activation by binding to CD44s protein and inducing lipid raft clustering.     

Glycine-extended gastrin induces matrix metalloproteinase-1- and -3-mediated invasion of human colon cancer cells through type I collagen gel and Matrigel

The effects of glycine-extended gastrin (G-Gly) on the invasion by colon cancer cells through stromal extracellular matrix and the role of metalloproteinases (MMPs) in this invasion were investigated. We found that 10(-9)-10(-6) M G-Gly significantly increased the invasiveness of 2 human colon cancer cell lines, LoVo and HT-29, both expressing the G-Gly-specific binding site but little gastrin/CCK-B receptor (gastrin receptor). LoVo cells expressed MMP-1, -2, -3 and -9. An amount of 10(-7) M G-Gly enhanced collagenase MMP-1 expression. Overexpression of enhanced green fluorescent protein (EGFP)-fused MMP-1 in LoVo cells, by cDNA transfection, enhanced invasiveness through type I collagen gel. Immunofluorescence study revealed that G-Gly increased the number of cytoplasmic vesicles containing MMP-1, some vesicles being released from the cells. The MMP-1 vesicles contained one of the ubiquitous coat proteins, Golgi-localized, gamma-adaptin ear-containing, ARF-binding proteins-2 (GGA-2). MMP-1 also colocalized with CD147 (EMMPRIN, an extracellular matrix metalloproteinase inducer in adjacent stromal cells). It was suggested that G-Gly increased the number of vesicles containing MMP-1 and that MMP-1 interacted with CD147 to increase invasion. G-Gly significantly enhanced the production of MMP-3, an activator of MMP-1 and -9, as well as gelatinase MMP-9 activity. The G-Gly-mediated MMP-9 increase was inhibited by treatment with anti-MMP-3 IgG and MMP-3 siRNA. Furthermore, G-Gly increased the proMMP-2 level, although no activated MMP-2 was found in conditioned medium in either the presence or the absence of G-Gly. By contrast, gastrin (10(-7) M) had no effect on the levels of these MMPs or the invasiveness of colon cancer cells in type I collagen gel and Matrigel. These effects of G-Gly on the activity and expression of MMPs and the invasiveness of colon cancer cells were inhibited by treating the cells with a broad-spectrum metalloproteinase inhibitor (CGS27023A) and nonselective gastrin/CCK receptor antagonists (proglumide and benzotript). But a gastrin/CCK-B receptor antagonist (YM022) did not inhibit the increased invasion by G-Gly. Together, these results demonstrate that G-Gly renders colon cancer cells more invasive by increasing MMP-1 and MMP-3 expressions via the putative G-Gly receptor and would thus be a good molecular target in a clinical setting.     

Comparison of the prognostic potential of hyaluronic acid, hyaluronidase (HYAL-1), CD44v6 and microvessel density for prostate cancer

Despite the development of nomograms designed to evaluate a prostate cancer (PCa) patient's prognosis, the information has been limited to PSA, clinical stage, Gleason score and tumor volume estimates. We compared the prognostic potential of 4 histologic markers, hyaluronic acid (HA), HYAL-1-type hyaluronidase (HAase), CD44v6 and microvessel density (MVD) using immunohistochemistry. HA is a glycosaminoglycan that promotes tumor metastasis. CD44 glycoproteins serve as cell surface receptors for HA, and the CD44v6 isoform is associated with tumor metastasis. HYAL-1-type HAase is expressed in tumor cells and, like other HAases, degrades HA into angiogenic fragments. Archival PCa specimens (n=66) were obtained from patients who underwent radical prostatectomy for clinically localized PCa and had a minimum follow-up of 72 months (range 72-131 months, mean 103 months). For HA, HYAL-1 and CD44v6 staining and MVD determination, a biotinylated HA-binding protein, an anti-HYAL-1 IgG, an anti-CD44v6 IgG and an anti-CD34 IgG were used, respectively. HA and HYAL-1 staining was classified as either low- or high-grade. CD44v6 staining and MVD were evaluated quantitatively and then grouped as either low- or high-grade. Using 72 months as the cut-off limit for evaluating biochemical recurrence, HA, HYAL-1, combined HA-HYAL-1, CD44v6 and MVD staining predicted progression with 96%, 84%, 84%, 68% and 76% sensitivity, respectively. Specificity was, 61% (HA), 80.5% (HYAL-1), 87.8% (HA-HYAL-1), 56.1% (CD44v6) and 61% (MVD). Sensitivity and specificity values for each marker did not change significantly in a subset of 45 patients for whom follow-up of longer than 112 months was available. In univariate analysis using the Cox proportional hazards model, preoperative PSA, Gleason sum, margin status, seminal vesicle, extraprostatic extension (EPE), HA, HYAL-1, HA-HYAL-1 and MVD, but not CD44v6, age and clinical stage, were significant in predicting biochemical recurrence (p < 0.05). In multivariate analysis using stepwise selection, only preoperative PSA (hazard ratio/unit PSA change=1.086, p < 0.0001), EPE (hazard ratio=6.22, p=0.0016) and HYAL-1 (hazard ratio=8.196, p=0.0009)/HA-HYAL-1 (hazard ratio=5.191, p=0.0021) were independent predictors of biochemical recurrence. HA was an independent predictor of prognosis if HYAL-1 staining inference was not included in the multivariate model. In our retrospective study with 72- to 131-month follow-up, EPE, preoperative PSA and HYAL-1 either alone or together with HA (i.e., combined HA-HYAL-1) were independent prognostic indicators for PCa.     

Transforming growth factor‐β induces CD44 cleavage that promotes migration of MDA‐MB‐435s cells through the up‐regulation of membrane type 1‐matrix metalloproteinase

CD44, a transmembrane receptor for hyaluronic acid, is implicated in various adhesion‐dependent cellular processes, including cell migration, tumor cell metastasis and invasion. Recent studies demonstrated that CD44 expressed in cancer cells can be proteolytically cleaved at the ectodomain by membrane type 1‐matrix metalloproteinase (MT1‐MMP) to form soluble CD44 and that CD44 cleavage plays a critical role in cancer cell migration. Here, we show that transforming growth factor‐β (TGF‐β), a multifunctional cytokine involved in cell proliferation, differentiation, migration and pathological processes, induces MT1‐MMP expression in MDA‐MB‐435s cells. TGF‐β‐induced MT1‐MMP expression was blocked by the specific extracellular regulated kinase‐1/2 (ERK1/2) inhibitor PD98059 and the specific phosphoinositide 3‐OH kinase (PI3K) inhibitor LY294002. In addition, treatment with SP600125, an inhibitor for c‐Jun NH₂‐terminal kinase (JNK), resulted in a significant inhibition of MT1‐MMP production. These data suggest that ERK1/2, PI3K, and JNK likely play a role in TGF‐β‐induced MT1‐MMP expression. Interestingly, treatment of MDA‐MB‐435s cells with TGF‐β resulted in a colocalization of MT1‐MMP and CD44 in the cell membrane and in an increased level of soluble CD44. Using an electric cell‐substrate impedance sensing cell‐electrode system, we demonstrated that TGF‐β treatment promotes MDA‐MB‐435s cell migration, involving MT1‐MMP‐mediated CD44 cleavage. MT1‐MMP siRNA transfection‐inhibited TGF‐β‐induced cancer cell transendothelial migration. Thus, this study contributes to our understanding of molecular mechanisms that play a critical role in tumor cell invasion and metastasis. © 2009 Wiley‐Liss, Inc.     

Regulation of CD44 expression and focal adhesion by Golgi phosphatidylinositol 4‐phosphate in breast cancer

CD44, a transmembrane receptor, is expressed in the standard or variant form and plays a critical role in tumor progression and metastasis. This protein regulates cell adhesion and migration in breast cancer cells. We previously reported that phosphatidylinositol‐4‐phosphate (PI(4)P) at the Golgi regulates cell migration and invasion in breast cancer cell lines. In this study, we showed that an increase in PI(4)P levels at the Golgi by knockdown of PI(4)P phosphatase SAC1 increased the expression of standard CD44, variant CD44, and ezrin/radixin phosphorylation and enhanced the formation of focal adhesions mediated by CD44 and ezrin/radixin in MCF7 and SK‐BR‐3 cells. In contrast, knockdown of PI 4‐kinase IIIβ in highly invasive MDA‐MB‐231 cells decreased these factors. These results suggest that SAC1 expression and PI(4)P at the Golgi are important in tumor progression and metastasis and are potential prognostic markers of breast cancers.     

A mesenchymal‐like phenotype and expression of CD44 predict lack of apoptotic response to sorafenib in liver tumor cells

The multikinase inhibitor sorafenib is the only effective drug in advanced cases of hepatocellular carcinoma (HCC). However, response differs among patients and effectiveness only implies a delay. We have recently described that sorafenib sensitizes HCC cells to apoptosis. In this work, we have explored the response to this drug of six different liver tumor cell lines to define a phenotypic signature that may predict lack of response in HCC patients. Results have indicated that liver tumor cells that show a mesenchymal‐like phenotype, resistance to the suppressor effects of transforming growth factor beta (TGF‐β) and high expression of the stem cell marker CD44 were refractory to sorafenib‐induced cell death in in vitro studies, which correlated with lack of response to sorafenib in nude mice xenograft models of human HCC. In contrast, epithelial‐like cells expressing the stem‐related proteins EpCAM or CD133 were sensitive to sorafenib‐induced apoptosis both in vitro and in vivo. A cross‐talk between the TGF‐β pathway and the acquisition of a mesenchymal‐like phenotype with up‐regulation of CD44 expression was found in the HCC cell lines. Targeted CD44 knock‐down in the mesenchymal‐like cells indicated that CD44 plays an active role in protecting HCC cells from sorafenib‐induced apoptosis. However, CD44 effect requires a TGF‐β‐induced mesenchymal background, since the only overexpression of CD44 in epithelial‐like HCC cells is not sufficient to impair sorafenib‐induced cell death. In conclusion, a mesenchymal profile and expression of CD44, linked to activation of the TGF‐β pathway, may predict lack of response to sorafenib in HCC patients.     

Tunicamycin inhibits cell proliferation and migration in hepatocellular carcinoma through suppression of CD44s and the ERK1/2 pathway

Tunicamycin (TM) is an N‐linked glycosylation (NLG) inhibitor with strong antitumor activity, the exact underlying molecular mechanism of which remains to be elucidated. In our previous studies, we found that TM reversed drug resistance and improved the efficacy of combination treatments for hepatocellular carcinomas (HCC). Here, we investigated the effects of TM on HCC cell proliferation and migration as well as the mechanism of those effects. Our results showed that TM inhibited cell proliferation and migration as well as induced apoptosis of hepatocellular carcinoma cells. TM inhibited proliferation of HCC cells by inducing cell apoptosis and cell cycle arrest at the G2/M phase. Meanwhile, TM inhibited migration of HCC cells by suppressing CD44s‐mediated epithelial‐mesenchymal transition (EMT). TM inhibited migration and invasion of HCC cells by decreasing CD44 expression and altering its glycosylation. In addition, CD44s is involved in promoting EMT and is associated with a poor prognosis in HCC patients. Overexpression of CD44s promoted tumor migration and activated phosphorylation of ERK1/2 in HCC cells, whereas TM inhibited CD44s overexpression‐associated cell migration. The ability of TM to inhibit cell migration and invasion was enhanced or reversed in CD44s knockdown cells and cells overexpressing CD44s, respectively. The MEK/ERK inhibitor U0126 and TM inhibited hyaluronic acid‐induced cell migration in HCC cells. Furthermore, TM inhibited exogenous transforming growth factor beta (TGF‐β)‐mediated EMT by an ERK1/2‐dependent mechanism and restored the TGF‐β‐mediated loss of E‐cadherin. In summary, our study provides evidence that TM inhibits proliferation and migration of HCC cells through inhibition of CD44s and the ERK1/2 signaling pathway.     

CD44 stimulation by fragmented hyaluronic acid induces upregulation of urokinase-type plasminogen activator and its receptor and subsequently facilitates invasion of human chondrosarcoma cells

It has been established that fragmented hyaluronic acid (HA), but not native high molecular weight HA, can induce angiogenesis, cell proliferation and migration. We have studied the outside-in signal transduction pathways responsible for fragmented HA-mediated cancer cell invasion. In our study, we have studied the effects of CD44 stimulation by ligation with HA upon the expression of matrix metalloproteinases (MMPs)-2 and -9 as well as urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1) and the subsequent induction of invasion of human chondrosarcoma cell line HCS-2/8. Our study indicates that (i) CD44 stimulation by fragmented HA upregulates expression of uPA and uPAR mRNA and protein but does not affect MMPs secretion or PAI-1 mRNA expression; (ii) the effects of HA fragments are critically HA size dependent: high molecular weight HA is inactive, but lower molecular weight fragmented HA (Mr 3.5 kDa) is active; (iii) cells can bind avidly Mr 3.5 kDa fragmented HA through a CD44 molecule, whereas cells do not effectively bind higher Mr HA; (iv) a fragmented HA induces phosphorylation of MAP kinase proteins (MEK1/2, ERK1/2 and c-Jun) within 30 min; (v) CD44 is critical for the response (activation of MAP kinase and upregulation of uPA and uPAR expression); and (vi) cell invasion induced by CD44 stimulation with a fragmented HA is inhibited by anti-CD44 mAb, MAP kinase inhibitors, neutralizing anti-uPAR pAb, anti-catalytic anti-uPA mAb or amiloride. Therefore, our study represents the first report that CD44 stimulation induced by a fragmented HA results in activation of MAP kinase and, subsequently, enhances uPA and uPAR expression and facilitates invasion of human chondrosarcoma cells.     

Expression of TNF‐α and CD44 is implicated in poor prognosis,cancer cell invasion,metastasis and resistance to the sunitinib treatment in clear cell renal cell carcinomas

Tumor necrosis factor‐α (TNF‐α) is involved in epithelial‐mesenchymal transition (EMT) and expression of CD44, a cancer stem cell marker, in several cancers. This study was performed to clarify the significance of TNF‐α and CD44 in clear cell renal cell carcinomas (ccRCCs). Expression of TNF‐α and CD44 was examined by immunohistochemistry in 120 ccRCCs. Involvement of TNF‐α in EMT and induction of CD44 was analyzed by monitoring expression of EMT‐related genes and CD44, and invasion in cultured ccRCC cell lines. TNF‐α and CD44 were immunolocalized mainly to carcinoma cells of high‐grade ccRCCs with positive correlations with primary tumor stage. A positive correlation was also obtained between TNF‐α and CD44 expression, and co‐upregulation of TNF‐α and CD44 was associated with primary tumor stage, distant metastasis, and poor prognosis. TNF‐α enhanced migration and invasion of ccRCC cells together with down‐regulation of E‐cadherin expression and up‐regulation of matrix metalloproteinase 9 and CD44 expression. TNF‐α also up‐regulated the expression of TNF‐α itself in ccRCC cells. Among the 25 ccRCC patients treated with sunitinib for metastatic disease, high CD44 expression was associated with poor treatment outcome. Importantly, residual carcinoma cells in the sunitinib‐treated metastatic ccRCCs were strongly positive for CD44, and the CD44 expression was significantly higher in the tumors from the sunitinib‐treated patients than in those from untreated ones. Our data show that TNF‐α plays an important role in progression of ccRCCs by inducing EMT and CD44 expression, and suggest that CD44 induced by TNF‐α may be involved in the resistance to the sunitinib treatment.     

Expression of Bmi-1, P16, and CD44v6 in Uterine Cervical Carcinoma and Its Clinical Signi.cance

Objective Bmi-1, a putative proto-oncogene, is a core member of the polycomb gene family, which is expressed in many human tumors. The p16 protein negatively regulated cell proliferation, whereas CD44v...     

CD44 variant 9 is a potential biomarker of tumor initiating cells predicting survival outcome in hepatitis C virus‐positive patients with resected hepatocellular carcinoma

This study investigated whether the expression of CD44 variant 9 (CD44v9) might be a functional marker of tumor‐initiating stem‐like cells in primary hepatocellular carcinomas (HCCs) of hepatitis C virus (HCV)⁺ patients and provide an indicator of patient survival, as well as associated mechanisms. A total of 90 HCV⁺ HCC patients who underwent surgery from 2006 to 2011 were enrolled and monitored for 2–8 years. Expression of CD44v9 was validated immunohistochemically in all HCCs, followed by comparative proteome, survival, and clinicopathological analyses. CD44 variant 8–‐10 was further evaluated in diethylnitrosamine‐induced HCCs of C57Bl/6J mice. Focally localized CD44v⁺ cells with a membranous staining pattern were detected in human HCV⁺ and mouse HCCs. CD44v9⁺ cells of HCCs were predominantly negative for Ki67 and P‐p38, indicating decrease of cell proliferation in the CD44v9⁺ tumor cell population, likely to be related to suppression of intracellular oxidative stress due to activation of Nrf2‐mediated signaling, DNA repair, and inhibition of xenobiotic metabolism. CD44v9 IHC evaluation in 90 HCV⁺ HCC cases revealed that positive expression was significantly associated with poor overall and recurrence‐free survival, a younger age, poor histological differentiation of HCCs, and high alkaline phosphatase levels compared with patients with negative expression. CD44v9 is concluded to be a potential biomarker of tumor‐initiating stem‐like cells and a prognostic marker in HCV⁺ HCC patients associated with Nrf2‐mediated resistance to oxidative stress.     

Improved detection of melanoma antigen-specific T cells expressing low or high levels of CD8 by HLA-A2 tetramers presenting a Melan-A/Mart-1 peptide analogue.

MHC class I tetramers containing peptide epitopes are sensitive tools for detecting antigen-specific CD8(+) T-cell responses. We demonstrate here that binding of HLA-A2 tetramers to CD8(+) T cells specific for the melanoma-associated antigen Melan-A/MART-1 can be fine-tuned by altering either the bound peptide epitope or residues in the alpha 3 domain of HLA-A2, which is important for CD8 binding. Antigen-specific T cells expressing high levels of CD8 could be detected using HLA-A2 tetramers containing the peptide AAGIGILTV, an epitope which is naturally processed and presented from Melan-A/MART-1. In contrast, low CD8-expressing, antigen-specific T cells could be detected efficiently only by using a mutated HLA-A2 tetramer with an altered CD8 binding site or, less efficiently, using the wild-type HLA-A2 tetramer loaded with the peptide analogue ELAGIGILTV, which is superior in stimulating antigen-specific T-cell responses. Our results suggest ways to optimize the identification and expansion of antigen-specific T cells with different requirements for the costimulatory CD8 molecule in facilitating T-cell receptor binding to peptide variants.