Characterization of the interaction between heterodimeric αvß6 integrin and urokinase plasminogen activator receptor (uPAR) using functional proteomics.

Urokinase plasminogen activator receptor (uPAR) and the epithelial integrin αvß6 are thought to individually play critical roles in cancer metastasis. These observations have been highlighted by the recent discovery (by proteomics) of an interaction between these two molecules, which are also both implicated in the epithelial-mesenchymal transition (EMT) that facilitates escape of cells from tissue barriers and is a common signature of cancer metastases. In this study, orthogonal in cellulo and in vitro functional proteomic approaches were used to better characterize the uPAR·αvß6 interaction. Proximity ligation assays (PLA) confirmed the uPAR·αvß6 interaction on OVCA429 (ovarian cancer line) and four different colon cancer cell lines including positive controls in cells with de novo ß6 subunit expression. PLA studies were then validated using peptide arrays, which also identified potential physical sites of uPAR interaction with αvß6, as well as verifying interactions with other known uPAR ligands (e.g., uPA, vitronectin) and individual integrin subunits (i.e., αv, ß1, ß3, and ß6 alone). Our data suggest that interaction with uPAR requires expression of the complete αß heterodimer (e.g., αvß6), not individual subunits (i.e., αv, ß1, ß3, or ß6). Finally, using in silico structural analyses in concert with these functional proteomics studies, we propose and demonstrate that the most likely unique sites of interaction between αvß6 and uPAR are located in uPAR domains II and III.

An immunomodulating peptide with potential to suppress tumour growth and autoimmunity.

Cancers and autoimmune diseases commonly co-exist and immune checkpoint inhibitor therapy (ICI) exacerbates autoimmune pathologies. We recently described a lipidic peptide, designated IK14004, that promotes expansion of immunosuppressive T regulatory (Treg) cells and uncouples interleukin-2 from interferon-gamma production while activating CD8+ T cells. Herein, we report IK14004-mediated inhibition of Lewis lung cancer (LLC) growth and re-invigoration of splenocyte-derived exhausted CD4+ T cells. In human immune cells from healthy donors, IK14004 modulates expression of the T cell receptor α/ß subunits, induces Type I IFN expression, stimulates natural killer (NK) cells to express NKG2D/NKp44 receptors and enhances K562 cytotoxicity. In both T and NK cells, IK14004 alters the IL-12 receptor ß1/ß2 chain ratio to favour IL-12p70 binding. Taken together, this novel peptide offers an opportunity to gain further insight into the complexity of ICI immunotherapy so that autoimmune responses may be minimised without promoting tumour evasion from the immune system.

An immunomodulating peptide to counteract solar radiation-induced immunosuppression and DNA damage.

Ultraviolet radiation (UVR) induces immunosuppression and DNA damage, both of which contribute to the rising global incidence of skin cancer including melanoma. Nucleotide excision repair, which is activated upon UVR-induced DNA damage, is linked to expression of interleukin-12 (IL-12) which serves to limit immunosuppression and augment the DNA repair process. Herein, we report an immunomodulating peptide, designated IK14800, that not only elicits secretion of IL-12, interleukin-2 (IL-2) and interferon-gamma (IFN-γ) but also reduces DNA damage in the skin following exposure to UVR. Combined with re-invigoration of exhausted CD4+ T cells, inhibition of UVR-induced MMP-1 release and suppression of B16F10 melanoma metastases, IK14800 offers an opportunity to gain further insight into mechanisms underlying the development and progression of skin cancers.

A novel lipidic peptide with potential to promote balanced effector-regulatory T cell responses.

T cell-dendritic cell (DC) interactions contribute to reciprocal stimulation leading to DC maturation that results in production of interleukin-12 (IL-12) and interferon-gamma (IFN-γ). Both cytokines have been implicated in autoimmune diseases while being necessary for effective immune responses against foreign antigens. We describe a lipidic peptide, designated IK14004, that modifies crosstalk between T cells and DCs resulting in suppression of IL-12p40/IFN-γ production. T cell production of interleukin-2 (IL-2) and IFN-γ is uncoupled and IL-12p70 production is enhanced. IK14004 induces expression of activating co-receptors in CD8+ T cells and increases the proportion of Foxp3-expressing CD4+ T regulatory cells. The potential for IK14004 to impact on signalling pathways required to achieve a balanced immune response upon stimulation of DCs and T cells is highlighted. This novel compound provides an opportunity to gain further insights into the complexity of T cell-DC interactions relevant to autoimmunity associated with malignancies and may have therapeutic benefit.

Norcantharidin induces HT-29 colon cancer cell apoptosis through the alphavbeta6-extracellular signal-related kinase signaling pathway.

Norcantharidin has been used as an efficacious anticancer drug in China for many years, but its true mechanism remains poorly understood. Intriguingly, in an in vitro series study of anticancer drugs, we found that norcantharidin can effectively inhibit epithelial tumor cells from expressing integrin alphavbeta6. Our previous studies have confirmed that integrin alphavbeta6 is closely relevant to malignant epithelial cell tumor biology behavior, and it can promote cancer cells to invade and metastasize through a special alphavbeta6-extracellular signal-related kinase (ERK) direct signaling pathway. In this study, we investigated the relationship between the norcantharidin anticancer mechanism and integrin alphavbeta6. After HT-29 colon cancer cells were treated with norcantharidin, cell apoptosis increased remarkably. The expression of alphavbeta6 and the amount of p-ERK decreased substantially; simultaneously, the linkage between alphavbeta6 and ERK was barely detectable. However, the expression of other integrins and the levels of mitogen-activated protein kinase hardly changed. On these grounds, we presumed that norcantharidin induced HT-29 colon cancer cell apoptosis through the alphavbeta6-ERK signaling pathway. This finding elicited a novel strategy for targeting the whole alphavbeta6-ERK signal pathway, rather than simply blocking the combining site of alphavbeta6-ERK in colon cancer treatment.

Suppression of integrin alphaupsilonbeta6 by RNA interference in colon cancer cells inhibits extracellular matrix degradation through the MAPK pathway.

Integrin alphaupsilonbeta6 plays a very important role in the progression of colon cancer cells and is now defined as a novel, independent prognostic indicator for aggressive colon cancer in humans. Herein, we use the RNA interfering technology to downregulate the expression of alphaupsilonbeta6 in colon cancer cells. Our data demonstrate that plasmid vector based shRNA can effectively down-regulate alphaupsilonbeta6 expression in protein and mRNA levels. Supression of integrin alphaupsilonbeta6 inhibits the phosphorylation and nonphosphorylation level of ERK1/2, the secretion of uPA, pro-MMP-9 and pro-MMP-2 in tumor conditioned medium, and more important, inhibits MAPK-dependent [(3)H] labeled collagen IV degradation via the plasminogen activation cascade. Our study demonstrates in vitro that supression of integrin alphaupsilonbeta6 inhibits extracellular matrix degradation through the MAPK pathway.

Integrin αvß6 plays a bi-directional regulation role between colon cancer cells and cancer-associated fibroblasts.

Tumor microenvironment (TME) is the cellular environment in which tumor exists, and it contributes to tumor formation and progression. The TME is composed of tumor cells, stromal cells, cytokines, and chemotactic factors of which fibroblasts are the main cellular components. In our present study, we found that colorectal cancer (CRC) cells expressing integrin αvß6 clearly could induce morphological changes in inactive fibroblasts and increased the expression of activated fibroblast markers such as α-smooth muscle actin (α-SMA) and fibroblast-activating protein (FAP). Those activated fibroblasts in the TME are called cancer-associated fibroblasts (CAFs). In order to investigate the mechanism by which CRC cells expressing integrin αvß6 activated CAFs, a series of assays have been carried out in the follow-up. We found that CRC cells could secrete inactive transforming growth factor ß (TGF-ß); however, integrin αvß6 activated TGF-ß, which subsequently activated fibroblasts. This process was disrupted by knockdown of integrin αvß6. In contrast, activated fibroblasts could promote CRC cell invasion. In particular, the strengthening effect on expression of integrin αvß6 in colon cancer cells was obvious. Additionally, we found that CAFs could secrete stromal cell-derived factor-1 (SDF-1) and promote CRC cell metastasis in distant organs via the SDF-1/C-X-C chemokine receptor type 4 (CXCR4) axis. Taken together, we assumed that CRC cells and CAFs activated one another and worked together to promote cancer progression, with integrin αvß6 playing a role in the bi-directional regulation of these cells. Hence, integrin αvß6 may serve as a therapeutic target for the future CRC treatment.

Norcantharidin Suppresses Colon Cancer Cell Epithelial-Mesenchymal Transition by Inhibiting the αvß6-ERK-Ets1 Signaling Pathway.

Norcantharidin (NCTD) is an efficacious anti-cancer drug that has been used in China for many years, but its underlying mechanism of action is still not fully understood. In the present study, we found that NCTD could induce morphological changes in colon cancer cells, causing a transition from a spindle-shaped morphology to a typical round or oval shape, which was indicative of a mesenchymal-epithelial transition (MET) process. Next, we investigated the mechanism by which NCTD induced the MET process. Using a transwell assay, we found that NCTD could suppress the migratory and invasive ability of colon cancer cells in a dose-dependent manner. Moreover, NCTD suppressed the expression of integrin αvß6, MMP-3, and MMP-9 as well as the polymerization of F-actin, further supporting its suppressive effect on migratory and invasive ability. Furthermore, the expression of αvß6, N-cadherin, vimentin and phosphorylated ERK was decreased, while the expression of E-cadherin was up-regulated. We verified that phosphorylated Ets1 was down-regulated substantially after treatment with NCTD. Taken together, our data demonstrated that NCTD could inhibit the EMT process of colon cancer cells by inhibiting the αvß6-ERK-Ets1 signaling pathway. This study revealed part of the mechanism through which NCTD could reverse the EMT process in colon cancer.

Direct integrin alphavbeta6-ERK binding: implications for tumour growth.

Blockade of the mitogen-activated protein (MAP) kinase pathway suppresses growth of colon cancer in vivo. Here we demonstrate a direct link between the extracellular signal-regulated kinase ERK2 and the growth-promoting cell adhesion molecule, integrin alphavbeta6, in colon cancer cells. Down-regulation of beta6 integrin subunit expression inhibits tumour growth in vivo and MAP kinase activity in response to serum stimulation. In alphavbeta6-expressing cells ERK2 is bound only to the beta6 subunit. The increase in cytosolic MAP kinase activity upon epidermal growth factor stimulation is all accounted for by beta6-bound ERK. Deletion of the ERK2 binding site on the beta6 cytoplasmic domain inhibits tumour growth and leads to an association between ERK and the beta5 subunit. The physical interaction between integrin alphavbeta6 and ERK2 defines a novel paradigm of integrin-mediated signalling and provides a therapeutic target for cancer treatment.

Rectal cancer masquerading as an amoeboma: case report and review of the literature.

Despite the prevalence of amoebiasis in many parts of the world, amoebomas are relatively uncommon. Amoebomas within the colorectum are indistinguishable from carcinomas either macroscopically or by barium enema examination and the presence of both conditions is much rarer still. Herein, we describe such a case and review the possible reasons for their coexistence.

Integrin αvß6 and transcriptional factor Ets-1 act as prognostic indicators in colorectal cancer.

BACKGROUND: Both transcriptional factor Ets-1 and integrin αvß6 play an important role in the development and progression of cancer. The aim of our study was to investigate the expression of Integrin αvß6 and Ets-1, two proteins' correlation and their clinical significance in colorectal cancerous tissues. RESULTS: The specimens were arranged into microarray using the immunohistochemistry method to investigate the expression of integrin αvß6 and transcriptional factor Ets-1 in these tissues. Among the 158 tissue specimens, 36.07% were positive for αvß6 expression, and 57.59% were positive for Ets-1 expression. There were obvious statistical differences existed regarding differentiation, N stage, M stage and TNM stage between αvß6 and Ets-1 positively and negatively expressing tumors. The correlation analysis confirmed the expression of αvß6 and Ets-1 were positively correlated in colorectal cancer. The Kaplan-Meier survival analysis showed that patients who were both αvß6 and Ets-1 positive relapsed earlier than those who were both αvß6 and Ets-1 negative; and the former group had much shorter survival time than the latter. And Cox model indicated that αvß6 and Ets-1 were the independent prognostic factors (RR = 2.175, P = 0.012 and RR = 3.903, P < 0.001). CONCLUSIONS: The expression of αvß6 and Ets-1 were positively correlated, and their expression degrees were associated with the differentiation, N stage, M stage and TNM stage of the tumors. Hence, the combination of αvß6 and Ets-1 can be used as a prognostic marker in colorectal cancer, especially for the early stage.

PKC promotes the migration of colon cancer cells by regulating the internalization and recycling of integrin αvß6.

Recently published studies have suggested that integrin trafficking is necessary to support cell migration, but the role of internalization and recycling of integrin αvß6 in colon cancer cells remained unclear. In our study, we demonstrated the existence of the integrin cycle and found that inhibition of ERK2 phosphorylation by PD98059 or deletion of the ERK2 direct binding site on the ß6 cytoplasmic domain could interrupt the internalization of integrin αvß6, but had no effect on its recycling. Furthermore, integrin αvß6 trafficking played a key role in the migration of colon cancer cells towards fibronectin. Activation of PKC significantly accelerated the internalization and recycling of integrin αvß6, which could facilitate rapid redistribution of integrin αvß6 and increase cell motility. When colon cancer cells became crowded, the increase in αvß6 levels at the cell surface was not accompanied by a change in total αvß6 expression in cell lysates. This change may be due to a redistribution of αvß6 in cell microstructures and a rapid cellular response towards the demands of migration.

alpha(v)beta(6) Integrin expression in diseased and transplanted kidneys.

BACKGROUND: Integrins have been implicated in the pathogenesis of a diverse range of kidney diseases. Herein, we provide the first detailed description of an epithelial restricted integrin, alpha(v)beta(6), in kidney biopsies from patients suffering acute and chronic renal diseases and after transplantation. METHODS: Immunoperoxidase staining for beta(6) was performed on 267 selected biopsy specimens from native (N= 126) and transplanted kidneys (N= 141) and scored semiquantitatively. The site of beta(6) expression in tubules was determined using haematoxylin counterstaining and by colocalization with Tamm-Horsfall protein. Comparisons were made between subcategories of diseases of native kidneys and between "service" and "protocol" biopsies of transplanted kidneys. RESULTS: beta(6), when present, is largely confined to the distal tubules and collecting ducts, colocated with Tamm-Horsfall protein. When sparsely present, it was often restricted to the tubular segment associated with the juxtaglomerular apparatus. It was found in tubular cells shed into the urine. beta(6) was not expressed in thin membrane nephropathy, or in nonproliferative forms of glomerulonephritis, with the exception of focal and segmental glomerulosclerosis (FSGS). It was diffusely expressed where there was glomerular necrosis or thrombosis and in most forms of acute or chronic tubulointerstitial disease. beta(6) was diffusely up-regulated in allografts biopsied for delayed function, in almost all kidneys that have clinical or subclinical rejection episodes and was prominent in chronic allograft nephropathy. CONCLUSION: beta(6) integrin is not normally expressed in adult native or transplanted kidneys but is commonly up-regulated in the distal tubule in disease. Our descriptive study suggests that it is a molecule worthy of further study.

Integrin expression in colon cancer cells is regulated by the cytoplasmic domain of the beta6 integrin subunit.

We have previously reported that the alphavbeta6 integrin upregulates its own expression in a protein kinase C-dependent manner with increasing cell density. The wild-type beta6 integrin subunit has also been shown to promote tumour growth in vivo and its growth-enhancing effect is regulated by both a MAP kinase binding motif on beta6 and the 11 amino acid C-terminal cytoplasmic extension unique to the beta6 subunit. Herein, we show that the 11 amino acid cytoplasmic extension is essential for the cell density-dependent increase in beta6 expression and that the 11 amino acid tail exerts a dominant negative effect on cell density- and PKC-mediated beta5 expression in alphavbeta6-expressing colon cancer cells. Cells that express beta6 lacking the 11 amino acid tail respond to PKC simulation with increased expression of only the beta5 subunit as seen for cells that lack constitutive alphavbeta6 expression. In contrast, loss of the ERK binding site on beta6 markedly impairs cell density- and PKC-dependent expression of either beta6 or beta5 in the presence or absence of the 11 amino acid tail, respectively. Our findings suggest that in alphavbeta6-expressing cells, a hierarchy of kinase signalling cascades exists and that the beta6-ERK2 interaction dominates over PKC-mediated signalling pathways responsible for integrin upregulation with cell confluence. Given the dominance of the beta6-ERK2 interaction over PKC-mediated expression of both beta5 and beta6 integrin subunits, targeting the beta6-ERK2 interaction may prove useful as an anticancer strategy in colon cancer.