Protein Kinase C–Dependent Mobilization of the α6β4 Integrin from Hemidesmosomes and Its Association with Actin-Rich Cell Protrusions Drive the Chemotactic Migration of Carcinoma Cells

We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the α6β4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express α6β4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the α6β4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis (∼1 ng/ml), the α6β4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained α6β4 in association with F-actin. Importantly, we demonstrate that this mobilization of α6β4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-α and that it is associated with the phosphorylation of the β4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin–rich cell protrusions that mediate α6β4-dependent cell movement but also the disruption of α6β4-containing hemidesmosomes by protein kinase C

Tetraspanin CD151 plays a key role in skin squamous cell carcinoma

Here we provide the first evidence that tetraspanin CD151 can support de novo carcinogenesis. During two-stage mouse skin chemical carcinogenesis, CD151 reduces tumor lag time and increases incidence, multiplicity, size, and progression to malignant squamous cell carcinoma (SCC), while supporting both cell survival during tumor initiation and cell proliferation during the promotion phase. In human skin SCC, CD151 expression is selectively elevated compared to other skin cancer types. CD151 support of keratinocyte survival and proliferation may depend on activation of transcription factor STAT3, a regulator of cell proliferation and apoptosis. CD151 also supports PKCα-α6β4 integrin association and PKC-dependent β4 S1424 phosphorylation, while regulating α6β4 distribution. CD151-PKCα effects on integrin β4 phosphorylation and subcellular localization are consistent with epithelial disruption to a less polarized, more invasive state. CD151 ablation, while minimally affecting normal cell and normal mouse functions, markedly sensitized mouse skin and epidermoid cells to chemicals/drugs including DMBA (mutagen) and camptothecin (topoisomerase inhibitor), as well as to agents targeting EGFR, PKC, Jak2/Tyk2, and STAT3. Hence, CD151 ‘co-targeting’ may be therapeutically beneficial. These findings not only support CD151 as a potential tumor target, but also should apply to other cancers utilizing CD151-laminin-binding integrin complexes

The role of alpha 6 integrin and its ligand, laminin, in prostate cancer.

Laminin is a major component of basal lamina and binds to the cell largely through a6 integrin receptors. The continued production of laminin by a tumor cell could confer an advantage to further invasion. The endogenous production of laminin by human prostate tumor cell lines OU145 (tumorigenic) and LNCaP (non-tumorigenic) was analyzed and related to their tumorigenic potential. Both produced classical laminin and S-laminin. OU145 laminin chain ratio (A:B1:B2:S) was (1.8):(1.0):(2.5):(1.0) compared to LNCaP cells, which was (1.0):(0):(10.0):(2.5). LNCaP cells retained most of their laminin production and their secreted forms were hypoglycosylated. LNCaP cells bound little laminin to their surface. In contrast, tumorigenic OU145 cells secreted most of their laminin in mature forms which bound to their surface. These features of LNCaP cells could contribute to their low adhesiveness and non-tumorigenic phenotype. Cell adhesion to' the extracellular matrix is mediated largely by integrins and is an important component to accomplish invasion. Prostate tumor cell lines were analyzed for their integrin content and function and related to their tumorigenic potential. OU145 cells contained α6, α3, α5, av integrins. Except for α3, LNCaP cells expressed all integrins with a lower molecular weight. LNCaP cells attached to ECM substrates to a lesser extent than DU145 cells. LNCaP cell deficient production of integrins and low attachment could affect their tumorigenic potential. In contrast DU145 cells express normal integrins and laminin and are tumorigenic. The α6 integrin (laminin receptor) persists during prostate tumor progression. To examine the role of a6 integrin in invasion, DU145 cells were sorted to select high (DU-H) and low (DU-L) a6 integrin expressors. DU-H cells contained α6β1 and α6β4 heterodimers compared to DU-L that only contained a low levels of α6β4. DU-H cells were three times more mobile on laminin as compared to the low expressors. Adhesion and migration were inhibited with anti-α6 antibody. Cells were injected intraperitoneally into SCID mice to test their invasive potential. DU-H showed greater invasion than DU-L, with increased expression of α6 integrins at the invasion areas. These data suggest that α6 integrin expression may be advantageous for invading cells

The integrin alpha6beta4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures

Functional studies on the alpha6beta4 integrin have focused primarily on its role in the organization of hemidesmosomes, stable adhesive structures that associate with the intermediate filament cytoskeleton. In this study, we examined the function of the alpha6beta4 integrin in clone A cells, a colon carcinoma cell line that expresses alpha6beta4 but no alpha6beta1 integrin and exhibits dynamic adhesion and motility on laminin-1. Time-lapse videomicroscopy of clone A cells on laminin-1 revealed that their migration is characterized by filopodial extension and stabilization followed by lamellae that extend in the direction of stabilized filopodia. A function-blocking mAb specific for the alpha6beta4 integrin inhibited clone A migration on laminin-1. This mAb also inhibited filopodial formation and stabilization and lamella formation. Indirect immunofluorescence microscopy revealed that the alpha6beta4 integrin is localized as discrete clusters in filopodia, lamellae, and retraction fibers. Although beta1 integrins were also localized in the same structures, a spatial separation of these two integrin populations was evident. In filopodia and lamellae, a striking colocalization of the alpha6beta4 integrin and F-actin was seen. An association between alpha6beta4 and F-actin is supported by the fact that alpha6beta4 integrin and actin were released from clone A cells by treatment with the F-actin- severing protein gelsolin and that alpha6beta4 immunostaining at the marginal edges of clone A cells on laminin-1 was resistant to solubilization with Triton X-100. Cytokeratins were not observed in filopodia and lamellipodia. Moreover, alpha6beta4 was extracted from these marginal edges with a Tween-40/deoxycholate buffer that solubilizes the actin cytoskeleton but not cytokeratins. Three other carcinoma cell lines (MIP-101, CCL-228, and MDA-MB-231) exhibited alpha6beta4 colocalized with actin in filopodia and lamellae. Formation of lamellae in these cells was inhibited with an alpha6-specific antibody. Together, these results indicate that the alpha6beta4 integrin functions in carcinoma migration on laminin-1 through its ability to promote the formation and stabilization of actin-containing motility structures

The integrin alpha 6 beta 4 and the biology of carcinoma

The integrin family of adhesion receptors plays a major role in epithelial organization and function. Moreover, the altered expression and function of specific integrins most likely contributes significantly to carcinoma progression. The integrin alpha 6 beta 4, the focus of this review, is a receptor for several members of the laminin family and is preferentially expressed at the basal surface of most epithelia, where it contributes to basement membrane interactions. Mounting evidence suggests that the alpha 6 beta 4 integrin plays a key role in carcinoma cell biology. Several histopathological studies have established a correlation between alpha 6 beta 4 integrin expression and tumor progression. The importance of alpha 6 beta 4 expression in tumors in underscored by the findings that invading fronts of several carcinomas are enriched in the expression of alpha 6 beta 4 integrin ligands, such as laminin-1 and laminin-5. The participation of the alpha 6 beta 4 integrin in invasion is supported further by in vitro functional studies using carcinoma cells that have been transfected with the beta 4 cDNA. The mechanisms by which alpha 6 beta 4 contributes to tumor progression are probably related to its mechanical and signaling properties and are currently under intense study

Towards a mechanistic understanding of tumor invasion--lessons from the alpha6beta 4 integrin

This review explores the mechanistic basis of carcinoma migration and invasion by focusing on the contribution of integrins. Integrins are essential for invasion not only for their ability to mediate physical interactions with extracellular matrices, but also for their ability to regulate signaling pathways that control actin dynamics and cell movement, as well as for growth and survival. Our comments center on a unique member of the integrin family, the alpha 6 beta 4 integrin, which is a receptor for the laminin family of basement membrane components. Numerous studies have implicated this integrin in the invasion of solid tumors and have provided a rationale for studying the mechanistic basis of its contribution to the invasive process. Such studies have revealed novel insights into the mechanism of carcinoma invasion that involve both the dynamics of cell migration and signaling pathways that regulate this migration

Protein Kinase C-α Phosphorylation of Specific Serines in the Connecting Segment of the β4 Integrin Regulates the Dynamics of Type II Hemidesmosomes

Although the regulation of hemidesmosome dynamics during processes such as epithelial migration, wound healing, and carcinoma invasion is important, the mechanisms involved are poorly understood. The integrin α6β4 is an essential component of the hemidesmosome and a target of such regulation. Epidermal growth factor (EGF) can induce hemidesmosome disassembly by a mechanism that involves serine phosphorylation of the β4 integrin subunit. Using a combination of biochemical and mutational analyses, we demonstrate that EGF induces the phosphorylation of three specific serine residues (S(1356), S(1360), and S(1364)) located within the connecting segment of the β4 subunit and that phosphorylation on these residues accounts for the bulk of β4 phosphorylation stimulated by EGF. Importantly, phosphorylation of these serines is critical for the ability of EGF to disrupt hemidesmosomes. Using COS-7 cells, which assemble hemidesmosomes type II upon exogenous expression of the α6β4 integrin, we observed that expression of a β4 construct containing Ser→Ala mutations of S(1356), S(1360), and S(1364) reduced the ability of EGF to disrupt hemidesmosomes and that this effect appears to involve cooperation among these phosphorylation sites. Moreover, expression of Ser→Asp mutants that mimic constitutive phosphorylation reduced hemidesmosome formation. Protein kinase C-α (PKC-α) is the kinase responsible for phosphorylating at least two of these serines, based on in vitro kinase assays, peptide mapping, and mutational analysis. Together, these results highlight the importance of serine phosphorylation in regulating type II hemidesmosome disassembly, implicate a cluster of serine residues within the connecting segment of β4, and argue for a key role for PKC-α in regulating these structures

Intestinal Restitution: Progression of Actin Cytoskeleton Rearrangements and Integrin Function in a Model of Epithelial Wound Healing

Superficial injury involving the mucosa of the gastrointestinal tract heals by a process termed restitution that involves epithelial sheet movement into the damaged area. The forces that drive epithelial sheet movement are only partially understood, although it is known to involve changes in the morphology of cells bordering the damage, such as the formation of large, flat, cytoplasmic extensions termed lamellae. We investigated the mechanism of epithelial sheet movement by following the response of the actin cytoskeleton and specific integrins (α6β4, α6β1, and α3β1) to wounding. To model this event in vitro, monolayers of T84 cells, well-differentiated colon carcinoma cells, were damaged by aspiration and the ensuing response was analyzed by a combination of time-lapse video microscopy, fluorescence confocal microscopy and antibody inhibition assays. We show that wound healing begins with retraction of the monolayer. α6β4 integrin is localized on the basal surface in structures referred to as type II hemidesmosomes that persist throughout this early stage. We hypothesize that these structures adhere to the substrate and function to retard retraction. Once retraction ceases, the wound is contracted initially by actin purse strings and then lamellae. Purse strings and lamellae produce a pulling force on surrounding cells, inducing them to flatten into the wound. In the case of lamellae, we detected actin suspension cables that appear to transduce this pulling force. As marginal cells produce lamellae, their basal type II hemidesmosomes disappear and the α6 integrins appear evenly distributed over lamellae surfaces. Antibodies directed against the α6 subunit inhibit lamellae formation, indicating that redistribution of the α6 integrins may contribute to the protrusion of these structures. Antibodies directed against the α3β1 integrin also reduce the size and number of lamellae. This integrin’s contribution to lamellae extension is most likely related to its localization at the leading edge of emerging protrusions. In summary, wounds in epithelial sheets initially retract, and then are contracted by first an actin purse string and then lamellae, both of which serve to pull the surrounding cells into the denuded area. The α6 integrins, particularly α6β4, help contain retraction and both the α6 integrins and α3β1 integrin contribute to lamellae formation