Involvement of CD44 and its variant isoforms in membrane-cytoskeleton interaction,cell adhesion and tumor metastasis

Summary CD44s (standard form of CD44) is a transmembrane glycoprotein whose external domain displays extracellular matrix adhesion properties by binding both hyaluronic acid (HA) and collagen. The cytoplasmic domain of CD44s interacts with the cytoskeleton by binding directly to ankyrin. It has been shown that post-translational modifications, such as phosphorylation (by protein kinase C), acylation (by acyl-transferase) and GTP-binding enhance CD44's interaction with cytoskeletal proteins. Most importantly, the interaction between CD44s and the cytoskeletal protein, ankyrin, is required for the modulation of CD44s cell surface expression and its adhesion function.Recently, a number of tumor cells and tissues have been shown to express CD44 variant (CD44v) isoforms. Using RT-PCR and DNA sequence analyses, we have found that unique CD44 splice variant isoforms are expressed in both prostate and breast cancer cell lines and carcinomas. Most importantly, intracellular ankyrin is preferentially accumulated underneath the patched/capped structures of CD44 variant isoform in both breast and prostate cancer cells attached to HA-coated plates. We propose that selective expression of CD44v isoforms unique for certain metastatic carcinomas and their interaction with the cytoskeleton may play a pivotal role in regulating tumor cell behavior during tumor development and metastasis.     

Thrombospondin as a mediator of cancer cell adhesion in metastasis

Summary Thrombospondin (TSP) is a 450 kDa adhesive glycoprotein. It is present in high concentrations in the platelet -granule and can readily be secreted following platelet activation where local concentrations can be increased by 3–4 orders of magnitude. TSP is also synthesized by a variety of other cells and is incorporated into their extracellular matrix. TSP is a homotrimer with a number of functional domains, at least four of which might serve as receptor recognizing regions. The amino-terminal heparin binding domain interacts with heparin, other glycosaminoglycans and glycolipids and likely recognizes specific cell surface proteoglycans. The central disulfide cross-linked region, 210 kDa non-reduced and 70 kDa reduced, contains a peptide motif CSVTCG which is apparently responsible for binding to glycoprotein IV (CD36) with high affinity. Immediately adjacent to the calcium binding region of TSP, which undergoes considerable molecular relaxation in the absence of calcium, is an RGDA sequence. TSP has been demonstrated to bind to integrins of the _v3 and _IIb3 class. The carboxy-terminal region of TSP also contains at least one binding epitope for a cell receptor. There are 2 well characterized genes for TSP and truncated forms of TSP have been detected which have inhibitory effects on angiogenesis. Finally, TSP can interact with fibrinogen and fibronectin, perhaps on cellular surfaces, which might serve as secondary receptor-like mechanisms for TSP binding and subsequent mediation of cell adhesion.     

Serum levels of the soluble adhesion molecules in patients with malignant melanoma

The incidence of malignant melanoma has been steadily increasing over the past decades. CD 44 is a transmembrane glycoprotein which is implicated in a number of adhesive and migratory events. Downregulation of CD 44 is implicated in the metastatic process. P-Selectin is a member of the selectin family of cell surface molecules. The levels of P-Selectin in biological fluids may be elevated in subjects with a variety of pathological conditions. In malignant melanoma, elevation of the plasma level of soluble intercellular adhesion molecule-1 (sICAM-1) has been associated with a reduction in disease-free survival. This study was performed to investigate the differences in the serum concentrations of the adhesion molecules in patients with malignant melanoma.The study group consisted of 52 patients with malignant melanoma and 20 healthy subjects. No meaningful difference was observed for P-selectin and sICAM 1 levels. A statistically significant decrease was observed in the cancer patients for serum CD 44 levels.     

Adhesion mechanisms in lymphatic metastasis

The role of cellular adhesion in regional lymph node metastasis of solid tumors has been investigated. The data reviewed is based on studies in four different tumor models of human, rat and murine origin. An in vitro assay measuring tumor cell attachment to cryostat sections of normal peripheral lymph nodes, obtained from the species of tumor origin was used to compare the adhesion of tumor sublines with different metastatic potentials. A good correlation was found between tumor cell potential to metastasize to regional nodes and the adhesion to the sections in all models studied [1–3]. The adhesion of all tumor lines could be blocked by Arg-Gly-Asp containing peptides while pretreatment of the cells with antibodies to integrins implicated ₁ and ₃ receptor complexes in the adhesion. Ligand binding assays provided indirect evidence that the preferential attachment of the metastatic tumor lines to the frozen sections was mediated via extracellular matrix proteins such as fibronectin, vitronectin and type IV collagen. As these basement membrane proteins have been localized to the outer surfaces of reticular fibers [4] which are known to permeate the lymph node and trasverse the subcapsular sinus [5] it is postulated that tumor cell attachment to these fibers may facilitate and possibly be required for tumor cell retention and growth in the invaded regional nodes.     

Fibronectin and integrins in invasion and metastasis

Summary The adhesive glycoprotein fibronectin and integrin receptors appear to play important roles in the progression of metastatic disease. Fibronectin is a multifunctional extracellular glycoprotein that has at least two independent cell adhesion regions with different receptor specificities. The cell adhesive region in the central portion of fibronectin is comprised of at least two minimal amino acid sequences - an Arg-Gly-Asp (RGD) sequence and a Pro-His-Ser-Arg-Asn (PHSRN) sequence - which function in synergy. Another cell adhesive region is located near the carboxy-terminus in the alternatively spliced IIICS module. The critical minimal sequences for this region are Leu-Asp-Val (LDV) and Arg-Glu-Asp-Val (REDV) which function in an additive rather than synergistic fashion. Integrins are heterodimeric, transmembrane cell adhesion receptors for fibronectin and other extracellular matrix molecules. Several different integrins bind to fibronectin. The ₅₁ fibronectin-specific integrin binds to the central RGD/PHSRN site. The ₄₁ integrin binds to the IIICS site. Fibronectin-integrin interactions are important in tumor cell migration, invasion, and metastasis. In addition to promoting cell adhesion to the extracellular matrix, these proteins may also function in chemotaxis and control of proliferation. Peptide and antibody inhibitors of fibronectin and integrin functions have been shown to be effective inhibitors of metastasis, and are potentially important reagents for the study and control of cancer.     

CD44 variant expression and estrogen receptor status in breast cancer

To understand the relationship between CD44 gene expressionand an established variable associated with aggressive behaviourin human breast cancer, we have studied apanel of 6 breast cell lines and 40breast tumors selected primarily on the basis ofestrogen receptor (ER) status. CD44s (standard form) mRNAwas assessed by semi-quantitative RT-PCR, and CD44 variantsincorporating exon v7 or v10 were studied byRT-PCR and Southern blot. While CD44 expression wasnot influenced by estrogen in ER+ve MCF-7 cells,CD44s expression was slightly higher (up to 2fold) in ER–ve cells but there was amarked decrease in the range of CD44 variantsincorporating exons v7 or v10. In microdissected tumors,the levels of CD44s showed no correlation withER status but the pattern of expression oflarger forms of CD44 incorporating variant exons v7and v10 was significantly different (p=0.005and p=0.015, respectively) between ER+ve andER–ve tumors, reflecting the pattern seen in thecell lines. These findings suggest that the profileof CD44 expression in breast cancer may reflectcellular differentiation as indicated by the ER phenotype.The influence of these differences in CD44 expressionon the increased metastatic potential of ER negativebreast cancer remains to be determined.     

CD44V6表达与乳腺癌侵袭转移的关系

目的研究乳腺癌中CD44V6的表达及其与乳腺癌侵袭转移的关系。方法应用免疫组织化学和RT-PCR方法,检测30例乳腺癌和10例乳腺良性肿瘤组织中CD44V6表达。结果CD44V6在乳腺癌组织中的表达明显高于良性组织;CD44V6在Ⅲ～Ⅳ期乳腺癌中表达明显高于Ⅰ～Ⅱ期,有淋巴结转移乳腺癌组织中CD44V6表达显著高于无淋巴结转移者;CD44V6的表达与乳腺癌的病理类型无关。结论CD44V6的表达,有助于预测乳腺癌进展程度和对淋巴结转移的判断。     

Integrin expression in malignant melanoma

Invasion of melanoma cells into the underlying interstitial stromal matrix is the initial step for subsequent local and distant metastasis. The invading tumor cell must interact with the extracellular matrix during the early stages of invasion and later during penetration of lymphatic and blood vessels. This interaction with different types of extracellular matrix predicts that the invasive cell must possess surface adhesion receptors with diverse ligand specificities, including the capacity to bind different types of collagens and adhesive glycoproteins. Metastatic melanoma cells do in fact express multiple adhesion receptors, including several of the receptors from the integrin family of heterodimers. The integrin receptors can be either extremely specific for a single ligand or capable of binding multiple ligands. It is likely that the tumor cell's repertoire of adhesion receptors may influence not only its adhesive properties but its metastatic characteristics as well. There is evidence that normal melanocytes have an integrin profile distinct from that of melanoma cells. In particular, melanocytes adhere poorly to laminin while metastatic melanoma cells bind well to this ligand. This difference in adhesion between the two cell types appears to reflect the fact that melanoma cells express a melanoma-specific integrin (% MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqefm0B1jxALjhiov2D% aebbfv3ySLgzGueE0jxyaibaiGc9yrFr0xXdbba91rFfpec8Eeeu0x% Xdbba9frFj0-OqFfea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs% 0dXdbPYxe9vr0-vr0-vqpWqaaeaabiGaciaacaqabeaadaqaaqGaaO% qaaiaadggadaWgaaWcbaGaamODaaqabaGccqaHYoGydaWgaaWcbaGa% aG4maaqabaaaaa!40CF!\[a_7 \beta _1 \]) that binds laminin and is not detectable in normal melanocytes. The presence of increased laminin receptors and enhanced laminin binding in melanoma cells may contribute to the malignant phenotype.     

CD44V6、MMPs与肺癌转移的研究进展

 肺癌的侵袭和转移是个多步骤多基因参与的复杂过程，CD44V6是一种分布极为广泛的细胞表面跨膜糖蛋白，主要参与细胞与细胞、细胞与细胞外基质之间的特异性粘连过程。研究表明：CD44V6的高表达有助于肿瘤细胞获得转移潜能。MMPs是一大组金属离子依赖的蛋白酶，它能降解细胞外基质（extracellular matrix, ECM）中的各种蛋白成分。近年来的研究发现，MMPs在肿瘤侵袭转移中的作用，除了降解ECM以外，它还能促进肿瘤血管生成。联合检测这两个指标对判断肺癌的转移、病变后发展以及预后评估具有重要的意义。     

Different adhesion properties of highly and poorly metastatic HT-29 colon carcinoma cells with extracellular matrix components: role of integrin expression and cytoskeletal components.

Integrin-mediated tumour cell adhesion to extracellular matrix (ECM) components is an important step in the development of metastatic lesions. Thus, integrin expression and integrin-mediated adhesion of colon carcinoma cells to various ECM components was examined. Poorly (HT-29P) and highly (HT-29LMM) liver-metastatic colon carcinoma cells were used to study the rates of adhesion to collagen I (C I), collagen IV (C IV), laminin (LN), fibronectin (FN), or vitronectin (VN) in a static adhesion assay (10-120 min). Cells were untreated or treated with oligopeptides (RGD, GRGDS, YIGSR, RGES), anti-integrin antibodies, or colchicine, nocodazole, cycloheximide, acrylamide or cytochalasin D (to disrupt cytoskeletal structures). Both cell lines expressed similar patterns of integrin expression (alpha2, alpha3, ,alpha6, alphav, beta1, beta4, and beta5) by immunocytochemistry and immunoprecipitation. HT-29LMM cells showed significantly higher rates of adhesion to LN (P < 0.001) and FN (P < 0.001), but significantly poorer rates of adhesion to C I (P < 0.05) and C IV (P < 0.001) than HT-29P cells, respectively, adhesion to VN was insignificant. RGD and GRGDS inhibited HT-29LMM cell adhesion to FN only. Pretreatment with anti-beta, or anti-alpha2 integrin subunits suppressed adhesion to C I and C IV, and adhesion to LN was inhibited with anti-beta1 or anti-alpha6 integrin. Anti-beta1 or anti-alphav blocked adhesion to FN. Pretreatment of cells with cytochalasin D, cycloheximide or acrylamide inhibited adhesive interactions of both cell lines to the ECM components. In contrast, colchicine and nocodazole had no effect. The results demonstrate that adhesion of HT-29 cells to ECM is mediated, in part, by different integrins, depending on the substrate. Poorly and highly metastatic HT-29 cells possessed different patterns of adhesion to the various ECM substrates, but these differences were not due to different expression of integrin subunits. The results also suggested that the initial adhesion of poorly or highly metastatic HT-29 cells to ECM components requires, in part, the presence of native action and intermediate filaments, but not of microtubules. Thus the adhesion of tumour cells to ECM components may be dependent on signal transduction and assembly of microfilaments.     

CD44在肿瘤进展中的作用

CD44 has been the subject of extensive research because of its role in cancer and many physiological processes. Through binding to different ligands, CD44 can initiate a series of cascade. CD44 not only can promote tumorigenic and tumor metastasis, but also can suppress tumor growth and progression. In-depth study of CD44 and its role in signal pathway may provide a new path for cancer treatment.     

肺癌骨桥蛋白和CD44v6的表达与转移关系的研究

目的：探讨骨桥蛋白（osteopontin，OPN）和粘附分子CD44v6在肺癌组织中的表达及其与肺癌转移的关系。方法：应用免疫组织化学技术（SP）检测57倒肺癌组织中OPN与CD44v6的表达水平，并将结果与30例良性肺部疾病组织中OPN及CD44v6水平相比较。结果：肺癌组织中OPN和CD44v6阳性表达率分别为57．9％（33／57）和54．4％（31／57），显著高于良性肺部疾病（肺部炎性假瘤）组织（P〈0．05），阳性表达率分别为16．7％和10．0％，肺癌组织中OPN与CD44v6的表斌足正相关.Kendall’s tau—b-0.503，P〈0．05；OPN与CD44v6在发生淋巴结转移的肺癌组织中阳性表达率分别达71．1％（27／38）和65．8％（25／38），显著高于无淋巴结转移的肺癌组织中阳性表达率（P〈0．05）。结论：OPN、CD44v6可能参与了肺痛的发生、发展和转移，联合检测它们的表达可作为肺癌生物学行为的一项评估指标。     

CD44的表达与人类非小细胞肺癌淋巴转移相关性的初步研究

目的 研究ＣＤ４４蛋白在人非小细胞肺癌组织中的表达及其与非小细胞肺癌淋巴转移的关系。方法 应用ＳＯＰ免疫组化染色法对４４例非小细胞肺癌组织和１０例癌旁肺组织染色,光镜观察,并应用计算机图像分析仪进行胞浆光密度测量分析。结果 光镜观察：有淋巴结转移组的癌组织高表达率为７５％（１８／２４）,无淋巴结转移组的癌组织高表达率为３５％（７／２０）,癌旁肺组织高表达率为０（０／１０）,三组间差异非常显著（Ｐ〉     

Hyaluronate Receptors Mediating Glioma Cell Migration and Proliferation

The extracellular matrix (ECM) of the central nervous system (CNS) is enriched in hyaluronate (HA). Ubiquitous receptors for HA are CD44 and the Receptor for HA-Mediated Motility known as RHAMM. In the present study, we have investigated the potential role of CD44 and RHAMM in the migration and proliferation of human astrocytoma cells. HA-receptor expression in brain tumor cell lines and surgical specimens was determined by immunocytochemistry and western blot analyses. The ability of RHAMM to bind ligand was determined through cetylpyridinium chloride (CPC) precipitations of brain tumor lysates in HA-binding assays. The effects of HA, CD44 blocking antibodies, and RHAMM soluble peptide on astrocytoma cell growth and migration was determined using MTT and migration assays. Our results show that the expression of the HA-receptors, CD44, and RHAMM, is virtually ubiquitous amongst glioma cell lines, and glioma tumor specimens. There was a gradient of expression amongst gliomas with high grade gliomas expressing more RHAMM and CD44 than did lower grade lesions or did normal human astrocytes or non-neoplastic specimens of human brain. Specific RHAMM variants of 85- and 58-kDa size were shown to bind avidly to HA following CPC precipitations. RHAMM soluble peptide inhibited glioma cell line proliferation in a dose-dependent fashion. Finally, while anti-CD44 antibodies did not inhibit the migration of human glioma cells, soluble peptides directed at the HA-binding domain of RHAMM inhibited glioma migration both on and off an HA-based ECM. These data support the notion that HA-receptors contribute to brain tumor adhesion, proliferation, and migration, biological features which must be better understood before more effective treatment strategies for these tumors can be found.     

CD44 increases the efficiency of distant metastasis of breast cancer

Metastasis is the predominant cause of death from cancer yet we have few biomarkers to predict patients at increased risk of metastasis and are unable to effectively treat disseminated disease. Analysis of 448 primary breast tumors determined that expression of the hylauronan receptor CD44 associated with high grade (p = 0.046), ER- (p = 0.001) and PR-negative tumors (p = 0.029), and correlated with increased distant recurrence and reduced disease-free survival in patients with lymph-node positive or large tumors. To determine its functional role in distant metastasis, CD44 was knocked-down in MDA-MB-231 cells using two independent shRNA sequences. Loss of CD44 attenuated tumor cell adhesion to endothelial cells and reduced cell invasion but did not affect proliferation in vitro. To verify the importance of CD44 to post-intravasation events, tumor formation was assessed by quantitative in vivo imaging and post-mortem tissue analysis following an intra-cardiac injection of transfected cells. CD44 knock-down increased survival and decreased overall tumor burden at multiple sites, including the skeleton in vivo. We conclude that elevated CD44 expression on tumour cells within the systemic circulation increases the efficiency of post-intravasation events and distant metastasis in vivo, consistent with its association with increased distant recurrence and reduced disease-free survival in patients.