Tetraspanins and Tumor Progression

Abstract

Transmembrane protein tetraspanins either promote or suppress tumor invasion and metastasis. Their effects on tumor progression depend on the multimolecular transmembrane complex called tetraspanin-enriched microdomain (TEM) and are attributed to the alterations in the 1) motogenic and mitogenic behaviors and/or 2) microenvironmental interactions of tumor cells. As the modifiers of cell membrane structure and function, tetraspanins have emerged as diagnostic and prognostic markers and therapeutic targets for tumor progression.

Introduction

Tumor cells progress through multiple and orchestrated steps before the metastatic lesions are established in distant organs. Among the progression, cell motility-related metastatic steps include the invasion before intravasation, intravasation, extravasation, and invasion after extravasation. Success in these steps requires the coordination between tumor cell-cell adhesion, cell-matrix adhesion, matrix degradation, and cell migration. Notably, transmembrane proteins of the tetraspanin superfamily directly regulate all of these cellular events (1–5). Tetraspanin are the type III proteins that contain four membrane-spanning segments with conserved CCG motifs, transmembrane polar residues, and multiple acylation sites (Figure 1) and are widely expressed in all eukaryocytes (1–5). Importantly, tetraspanins regulate not only the motility-related behaviors of tumor cells but also the interaction between tumor cells and their microenvironment. For example, CD151 promotes the neovascularization in breast cancer xenografts (6), and Tspan8 or CO-029 elevates the angiogenesis of pancreatic cancer through secreted exosomes (7–8). Another characteristic related to tumor progression of tetraspanins is their ability to organize other transmembrane proteins such as cell adhesion molecules, growth factor receptors, and proteases into membrane complexes termed tetraspanin webs, glycosynapses, or tetraspanin-enriched microdomains (TEMs), with which intracellular signaling proteins such as PI4K, PKC, and syntenin are associated (3, 9–14). Upon partition into TEMs, the activity of these transmembrane proteins becomes altered, although the biochemical and biophysical mechanisms remain unclear. Of the 33 human tetraspanins, approximately half have been experimentally studied, and several have been shown to correlate with tumor patient prognosis and regulate tumor progression and metastasis. This review focuses on the roles and mechanisms of relatively better understood tetraspanins, i.e., CD9, CD63, CD82, CD151, and Tspan8, in tumor invasion and metastasis (Table 1).

Figure 1. Schematic depiction of tetraspanins and tetraspanin-enriched microdomain (TEM).

(A) Tetraspanins feature four transmembrane, two extracellular (EC1 and EC2), and an intracellular N-terminal, loop, and C-terminal domains. Most tetraspanins are glycosylated in either EC2 or EC1 domains. For example, CD63, CD82, CD151, and Tspan8 are glycosylated in EC2 while CD9 in EC1. The cysteine residues in EC2, typically in even number, form disulfide bonds, while the cysteine residues proximal to the interface between inner leaflet and cytosol are constantly palmitoylated. Characteristic strong polar residues are found in the transmembrane domains. Some tetraspanins such as CD63, CD82, CD151, and Tspan8 also contain “YXXΦ” sorting motif in C-terminus while others such as CD9 do not. Letters denote the code of amino acids. (B) TEMs are formed on the basis of dimeric tetraspanins, and these dimers further associate with each other to form multimeric tetraspanin complex in which cell adhesion proteins, growth factor receptors, proteases, and intracellular signaling proteins are sequestered. TEMs likely interact with a specific set of membrane lipid and alter the glycosylation of membrane molecules.

	Roles in tumor progression	Tumors involved	Associated proteins	Expression patterns in nontumor tissues	Possible mechanisms involved in tumor progression
Tspan 8 (CO-029)	Correlates	Colorectal, gastric, esophageal, pancreatic, and liver cancers	Tetraspanins CD9 and CD81 IgSF member FPRP EpCAM CD44 Integrins α6β4, α3β1, and α6β1 Intracellular protein PI4K.	Epithelial, endothelial, muscle, and nerve tissues	Facilitates metastasis by promoting angiogenesis.
CD151	Correlates	Breast, prostate, colon, liver, pancreas, lung, and gum cancers	Tetraspanins CD9, CD63, CD81, CD82, and Tspan 8 IgSF member EWI-2 EpCAM Integrins α3β1, α5β1, α6β1, and α6β4 Intracellular proteins PI4K and PKC	Epithelial, endothelial, platelets, and muscle tissues	Facilitates metastasis by promoting invasion, intravasation, and probably angiogenesis and by upregulating proteases responsible for degrading extracellular matrix.
CD9	Inversely correlates	Lung, breast, colon, skin, ovary, uterus, stomach, oral cavity, head and neck, prostate, and pancreas cancers and hematopoietic malignancy	Tetraspanins CD63, CD81, CD82, CD151, and Tspan8 IgSF members EWI-2, FPRP, and ICAM Integrins α2β1, α3β1, α5β1, and α6β1 Intracellular proteins PI4K and PKC	Epithelial, endothelial, muscle, nerve, and hematopoietic tissues	Downregulates the motility of tumor cells and growth factor signaling in tumor cells, regulates the activity of matrix-degrading enzymes, and forms a complex with podoplanin, which sensitizes tumor cells to be arrested in microcirculation or to be lysed by IL2-activated T and NK cells.
CD63	Probably inversely correlates	Melanoma, ovarian tumor, and lung cancer	Tetraspanins CD9, CD81, CD82, and CD151. Integrins α4β1, α3β1, α5β1, α6β1, and αIIbβ3. Intracellular proteins PI4K and syntenin-1	Ubiquitously expressed as a lysosome marker	Facilitates the transport of matrix-degrading proteins to lysosome to be degraded and upregulates the activity of MMP inhibitors.
CD82	Inversely correlates	Prostate, breast, lung, bladder, colon, pancreas, stomach, liver, oral, esophageal, penile, and endometrial cancers	Tetraspanins CD9, CD63, CD81, and CD151. IgSF member EWI-2 Integrins α3β1, α4β1, and α6β1	Epithelial, endothelial, and hematopoietic tissues	Downregulates the signaling pathways of its associated proteins such as cell adhesion molecules and growth factor receptors to inhibit cell motility, probably also alters the compartmentalization and cytoskeletal interaction of plasma membrane to inhibit cell motility, and interacts with DARC on endothelial cells to cause the senescence of tumor cells.

Tumor progression-promoting tetraspanins

Tspan8

Tspan8 or CO-029 was originally identified as a tumor-associated antigen (15), and its expression correlates with, or promotes, tumor progression (7, 16–18). The upregulation of Tspan8 expression is associated with the progression of pancreatic, hepatic, esophageal, gastric, and colorectal carcinomas, i.e., higher Tspan8 expressions are typically found in the more advanced stages of these tumors (15, 17–20). The occurrence and level of Tspan8 expression seem associated with the poor prognosis of gastrointestinal tumor patients.

The forced expression of Tspan8 in pancreatic and esophageal tumor cells promotes the metastasis to a distal organ like lung in animal spontaneous and/or experimental metastasis assays (16, 20). Tspan8 also facilitates the intrahepatic metastasis of hepatocellular tumor cells (21). It appears that tumor cells expressing Tspan8 prefer metastasizing without stopping in lymph nodes. In pancreatic tumor, Tspan8 expression leads to the hemorrhage around primary and metastatic tumors and disseminated intravascular coagulation (16)

The forced expression of Tspan8 results in ADAM12m-mediated enhancement of the migratory and invasive abilities of esophageal tumor cells (20). Notably, Tspan8 promotes tumor angiogenesis in pancreatic cancer animal models by enhancing endothelial cell migration and proliferation, capillary sprouting, and endothelial progenitor cell maturation (7–8). Through the tumor-secreted exosomes that are enriched with integrin α4 and VCAM-1, Tspan8-expressing tumor cells upregulate the expression of vascular endothelial growth factor (VEGF) and its receptor, chemokines and their receptors, von Willebrand factor, and Tspan8 itself in endothelial cells and VEGF, matrix matalloproteinases (MMPs), and urokinase plasminogen activator (UPA) in tumor-adjacent fibroblasts (7–8).

In addition to the classical constituents of TEM such as tetraspanins, integrins, and IgSF proteins, Tspan8-enriched microdomain also contains EpCAM, CD44v6, claudin 7, and C4.4A in colorectal cancer and pancreatic cells (16, 18, 22–23). Also in colorectal and pancreatic cancer cells, integrin α6β4 selectively associates and colocalizes with CD151 and Tspan8 and such coalescence is strengthened by protein kinase C (PKC) activation (19, 22).

CD151

CD151 expression is upregulated upon the progression and metastasis of various human solid malignant tumors such as breast, prostate, colon, liver, pancreas, and gum cancers, compared with that in corresponding normal tissues (6, 24–30). In general, higher CD151 expression correlates with a worse prognosis for cancer patients, i.e., lower survival and/or higher recurrence rates (6, 25–26, 28, 31–32).

In animal tumor models, CD151 promotes the distal metastasis of tumor by, at least, enhancing tumor cell intravasation (33) and tumor vascularization (6). CD151 monoclonal antibodies block the lung metastasis of epidermoid carcinoma, fibroblastoma, and colon adenocarcinoma in mice (34–35), while CD151 silencing reduces the lung metastasis of breast cancer in an experimental metastasis mouse model (36). In addition, CD151 silencing or overexpression significantly affects primary tumor growth in animals (6, 27–28). Notably, CD151 silencing sensitizes breast cancer cells to the ErbB2 antagonist treatment (37).

Direct enhancement of tumor cell movement appears to be the cellular mechanism for CD151 to promote tumor metastasis. In vitro, the overexpression of CD151 promotes tumor cell migration and invasion while the knockdown of CD151 usually inhibits them (27–28, 32, 35, 38–40). In vivo, a CD151 monoclonal antibody that blocks tumor metastasis inhibits the movement of tumor cells away from the primary tumor mass by attenuating the rear detachment from matrix of the invading tumor cells (33). Recent studies underline that CD151 expression in tumors correlates with the degree of tumor neovascularization and ability of tumor cells to induce angiogenesis (6, 32), implying that angiogenesis is involved in the prometastatic activity of CD151.

CD151 facilitates or stabilizes tumor cell-cell adhesion and strengthens tumor cell-laminin adhesion (38, 40–43). In addition, CD151 upregulates growth factor-induced signaling in tumor cells. CD151 expression is functionally associated with HGF/c-Met signaling in breast, liver, and salivary gland cancers (28, 39); and CD151 silencing specifically attenuates TGFβ1-induced p38 MAP kinase activation in MDA-MB-231 breast cancer cells (36). Moreover, CD151 enhances the pericellular proteolysis of tumor cells through association and activation of MMP7 and upregulation of MMP9 expression (28, 32, 35, 44). CD151-dependent pericellular proteolysis and -facilitated signaling likely contribute to its mediated promotion of tumor progression. However, it is unclear how CD151-dependent strengthening of cell-cell and cell-ECM adhesions leads to more progressive tumors. Presumably proper tumor cell-cell adhesion could accelerate collective movement of tumor cells when they depart from the primary tumor as a cohort or enhanced tumor cell adhesiveness facilitates the homing of metastatic tumor cells to the target tissue. It is also unclear how CD151-enhanced pericellular proteolysis reconciles with its reinforcement roles in cell adhesions.

Tumor progression-suppressing tetraspanins

CD9

CD9 is considered to function primarily as a progression and metastasis suppressor in solid tumors (5). Clinical and pathologic findings indicate that downregulation of CD9 correlates with the progression of malignant tumors from lung, breast, colon, skin, ovary, uterus, stomach, oral cavity, head and neck, prostate, pancrease, and several other organs and tissues (45–58). CD9 expression was also reported to correlate inversely with the progression of several hematopoietic malignancies (59–61). Reduced expression of CD9 is commonly observed in metastatic lesions compared with primary tumor, and patients with tumors lacking CD9 are typically at advanced stages. Although the prognostic importance of CD9 in the survival of tumor patients has not always been conclusive, most studies support the idea that CD9 expression in tumors correlates with better overall and/or disease-free survival rates, longer disease-free interval, and/or low recurrence rate (25, 49, 52, 55, 57, 62–66). In contrast, two studies on gastric cancer suggest that higher CD9 expression appears to be associated with more progressive tumors and poorer prognosis (67–68).

Studies with CD9 overexpression, silencing, and antibody treatment using spontaneous metastasis assay in orthotopic or ectopic tumor implantation models emphasize that CD9 suppresses the local lymph node and/or distal organ metastasis of various solid tumors in mouse (69–73). Because CD9 inhibits cell motility such as migration and invasion of neoplastic cell lines from lung, breast, skin, gastric, pancreatic, and bladder tumors in vitro (24, 69, 74–76), CD9 likely renders tumor cells more static in vivo, leading to the suppression of tumor metastasis. However, it was also reported that CD9 expression positively correlates with cell motility in some tumor cells. For example, the overexpression of CD9 in B16F1 and SbCl2 melanoma cell lines had increased capability to invade Matrigel (77), and CD9 silencing in MCF7 breast cancer cells reduces invasiveness (78). Such discrepancy reflects the complexity of CD9 function and may result from different lipid compositions such as GM3 content in the plasma membrane of different tumor cells (74, 76). In addition, CD9 may regulate tumor-endothelial cell interaction and tumor lymphangiogenesis because of its presence in lymphatic vessels (55–56, 79). Interestingly, the cell surface CD9 proteins reduce platelet aggregation activity and lung retention of HT1080 fibroblastoma cells by associating with podoplannin, a metastasis-promoting and platelet aggregation-inducing factor (80). CD9-podoplannin complex neutralizes podoplannin-mediated platelet aggregation, which facilitates the arrest of tumor cell emboli in the microcirculation and prevents tumor cells from immune assault in the circulation. Another observation related to immune assault is that CD9 overexpression sensitizes myeloma cells to the lysis mediated by interleukin 2-activated T and NK cells (81). Finally, the attenuated proliferation and survival of tumor cells could partially contribute to CD9-mediated suppression of tumor progression (73, 77, 82–84).

How CD9 inhibits tumor cell motility is not firmly established. The mechanistic analyses have focused on integrin-mediated cell-ECM adhesion based on knowledge of the CD9-integrin complex. But CD9 appears to regulate both cell-cell and -ECM adhesions, as well as adhesion-dependent cytoskeletal reorganization and cellular morphological changes (79, 83, 85–87). Also, CD9-containing TEMs associate and colocalize with MT1-MMP and modulate the proteolytic activities of MT1-MMP, MMP-2, and MMP-9 (78, 82, 85, 88). The reported effects of CD9 on cell adhesion and MMP activity, however, are diverse as the effect of CD9 on cell motility, and further clarifications are needed. Studies on CD9 signaling have provided additional or alternative insights into the mechanisms. For example, CD9 potentiates the juxtacrine activities of proHB-EGF and transmembrane TGFα but specifically attenuates EGFR signaling through the downregulation of surface expression of EGFR (53, 89, 90), suggesting that CD9 alters growth factor signaling in tumor cells. CD9-containing TEMs also alter PI3K/Akt and Wnt signaling (82, 85, 87). The conclusions from these signaling studies are consistent with the observations that CD9 affects not only cell motility but also cell survival and proliferation. In summary, it is relatively clear that CD9 suppresses cancer progression, but more studies are required to appreciate how CD9 expression and signaling contributes to the cancer phenotype.

CD63

The expression of CD63 is downregulated upon the progression of melanoma, ovarian tumor, and lung adenocarcinoma in some studies (91–94) but does not correlate with the progression of tumors from prostate, lung, thyroid, and pancreas in other studies (49, 95–97). Nevertheless, the subcutaneous injection of CD63-overexpressing melanoma cells into nude mice led to the decreased rate of tumor growth while the intravenous injection had the reduced numbers of peritoneal and subcutaneous metastases (98). Up- or downregulating cellular expression of CD63 in vitro revealed that CD63 inhibits tumor cell migration and invasion, reduces matrix degradation, and alters cell-matrix adhesion (99–100). Besides interacting with TEM, CD63 reduces MT1-MMP expression by driving MT1-MMP to lysosomes for degradation (101) and binds to TIMP-1, an inhibitor of MMPs, on the cell surface (102). Consistently, decreased CD63 expression leads to increased activity of MMP2 and MMP9 (100).

CD82

CD82 is widely expressed in epithelial tissues, but its expression is often reduced or lost upon tumor progression (103–108). CD82 level is inversely correlated with patient prognosis in a variety of solid tumors such as prostate, breast, colorectal, pancreatic, bladder, lung, oral, gastric, liver, esophageal, penile, and endometrial cancers (103, 109–115). Notably, in breast cancer, CD82 expression is preferentially lost in estrogen receptor-positive tumors but constantly found in the primary and metastatic lesions of estrogen receptor-negative ones (116–118)

In animal models, CD82 inhibits tumor metastasis, in the majority of reports, without affecting the proliferation of primary tumors (103–104, 106–107, 109, 111–113). The forced expression of CD82 in prostate, breast, and liver cancer cells, as well as melanoma and fibroblatoma cells, significantly suppressed metastasis in spontaneous and/or experimental metastasis assays (103, 111, 119–121). Direct administration of CD82-expressing viruses into pancreatic or lung cancer also suppressed metastasis in orthotopic tumor animal models (72, 122).

At the cellular level, CD82 directly inhibits tumor cell migration and/or invasion (106). In migrating cells, CD82 inhibits both protrusive and retraction cellular processes by deregulating actin organization (our unpublished data). CD82 also indirectly inhibits cell motility by elevating cell-cell adhesion (112). Recently, CD82 was found to directly bind to Duffy antigen receptor for chemokines (DARC) on endothelium at the intravasation step, thereby leading to the growth arrest and senescence of CD82-positive tumor cells. In contrast, CD82-negative tumor cells entered the circulation and subsequently established metastasis (123).

At the molecular level, CD82 alters the functions or activities of membrane molecules such as integrins, EGFR, c-Met, and uPAR, some of which associate with CD82 in TEMs, by modulating either their trafficking or membrane compartmentalization (124–127). Such effects likely result from the specific interaction of CD82 with membrane lipids such as GM2 and CD82-induced alteration of membrane lipid composition (128–130). Consequently, the downstream signaling derived from these membrane molecules such as src, p130^CAS/Crk, and Rho small GTPases are subsequently downregulated or deregulated by CD82 (126, 131–132) and lead to the perturbation of cytoskeletal rearrangements that are required for cell motility. It was also reported that CD82 expression reduced MMP9 enzymatic activity and increased TIMP1 level (133).

Concluding remarks

Being localized at cell-cell and -matrix contacts and associated with cell adhesion molecules such as IgSF proteins and integrins, tetraspanins regulate cell-cell and -matrix adhesion. Lines of evidence support the assumption that tetraspanins also modify growth factor signaling. Because cell adhesion molecules and growth factors directly regulate cell motility, tetraspanins basically modulate most motogenic signaling activities at or from the cell membrane. One emerging theme is that tetraspanins take an active part in membrane trafficking such as endocytosis and exocytosis (134). Another emerging theme is that tetraspanins are associated with the proteolysis at or near the plasma membrane by modulating the activities of various proteases such as MMP, MT1-MMP, ADAM, and γ-secretase (14, 135–137). Both themes provide additional dimensions of regulatory mechanisms for cell-cell adhesion, cell-matrix adhesion, and growth factor signaling. Based on their functional relationship with cell membrane, we postulate that, at the molecular level, tetraspanins are organizers for membrane proteins and lipids. Also, the signaling events associated with pro- or antitumor progression activity of a given tetraspanin are typically multifarious, implying that tetraspanins affect the global functions of, and at, the cell membrane.

At the cellular level, tetraspanins typically regulate tumor cell movement by altering cell-cell and -matrix interactions. Hence, whether epithelial-to-mesenchymal transition (EMT) or mesenchymal-to-epithelial transition (MET) is involved becomes an interesting question. It was reported that tetraspanin TM4SF5 promoted liver tumorigenesis through EMT (138). CD151, however, strengthened cell-cell adhesion despite enhancing tumor cell movement. Also, the genetic ablations of several tetraspanins that regulate tumor progression did not reveal altered EMT or MET during animal development though genetic compensation may exist. Hence, if EMT and/or MET are involved, they may not be the canonical ones, but they do deserve further study.

The functional difference between tetraspanins in tumor progression may result from the compositional difference of TEMs. The difference in TEM constituents leads to diversity of TEMs in membrane properties such as signaling, cytoskeletal connection, and/or curvature and subsequent difference in function. For example, CD82 and CD151 likely sequester and associate different proteins and lipids to form distinct TEMs. In addition, different abilities and behaviors of tetraspanins in trafficking may also contribute to their functional differences. For example, CD82 and CD151 exhibit significant differences in trafficking kinetics, route, and destination (130, our unpublished data). Such diverse behaviors in trafficking could result in different destiny of their associated cell adhesion proteins, growth factor receptors, and proteases and subsequently lead to opposite functions.

Tetraspanins discussed herein are the ideal diagnostic, staging, and prognostic markers and promising therapeutic targets for tumor progression. For example, the interference of coordinated activities of protease-tetraspanin complex may be a constructive therapeutic approach to limit tumor invasion and metastasis. Besides either promoting or suppressing tumor progression, tetraspanins are typically the wide-spectrum modifiers for the progression of various solid tumors, suggesting that the element(s) determining the activities of tetraspanins toward tumor progression lies within tetraspanin molecules per se. Identifying the structural element(s) could be therapeutically beneficial. However, the analyses of 1) structural similarities between promoters such as CD151 and Tspan8 or between suppressors such as CD9 and CD82 and 2) structural differences between promoters and suppressors have so far not resulted in meaningful insight. Although the aforementioned tetraspanins are not crucial for cell survival or proliferation, they alter the growth of primary tumor in animal model (6, 27). A few studies noted that the expression of tetraspanins such as CD151 and Tspan8 desensitized tumor cells to chemotherapy or apoptosis (23, 37). The mechanism is unclear but is probably unrelated to their association with integrins, because tumor cells typically survive and proliferate well in an anchorage- or integrin-independent manner. Compared to cell movement, the effect of tetraspanins on tumor growth and survival appears to be less prominent but nevertheless provides another advantage therapeutically. Together, more molecular and cellular information on tetraspanins needs to be obtained and deeper mechanistic insight on the roles of tetrapsanins in tumor progression needs to be gained before clinical applications of tetraspanins can be realized. Thus, in addition to delineating the mechanisms at the molecular, cellular, and organism levels and assessing the roles of “novel” tetraspanins in tumor progression, future studies should further explore the possibility of tetraspanins as diagnostic, staging, and prognostic markers and therapeutic targets for tumor progression.

Abbreviations:

DARC

Duffy antigen receptor for chemokines

ECM

extracellular matrix

EMT

epithelial-to-mesenchymal transition

KO

knock out

MET

mesenchymal-to-epithelial transition

MMP

matrix metalloproteinase

PI-3K

phosphatidylinositol-3 kinase

PI-4K

phosphatidylinositol-4 kinase

PKC

protein kinase C

TEM

tetraspanin-enriched microdomain

UPA

urokinase plasminogen activator

VEGF

vascular endothelial growth factor