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The tumor microenvironment dynamically regulates the progression of cancer. In the breast, a unique component of the microenvironment is the myoepithelial cell. Normal myoepithelial cells act as "natural tumor suppressors"; however, more recent evidence suggests that these cells develop phenotypic changes, which may contribute to loss of tumor suppressor activity. We have shown that myoepithelial cells in a subset of preinvasive ductal carcinoma in situ (DCIS) upregulate expression of the integrin αvβ6, switching on tumor promoter activity through activation of TGFβ and MMP9. This makes the tumor microenvironment more permissive to invasion, seen both in vitro and in vivo. In human tissue samples, increased myoepithelial αvβ6 expression correlated with increased risk of disease progression and recurrence. Current estimates suggest that as many as 50% of DCIS cases will never progress in the patient's lifetime, but there are no markers to predict the outcome of individual cases. The identification of αvβ6 in a subset of DCIS presents a unique way to stratify patients with DCIS into those who may or may not progress to more serious disease. As αvβ6 is not expressed on most normal adult tissues, this finding may also provide novel targets for therapy in this high-risk group., (©2014 American Association for Cancer Research.)

The cell surface receptor alpha(v)beta(6) is epithelial specific, and its expression is tightly regulated; it is low or undetectable in adult tissues but has been shown to be increased in many different cancers, including pancreatic, cervical, lung, and colon cancers. Studies have described alpha(v)beta(6) as a prognostic biomarker linked to poor survival. We have recently shown the feasibility of imaging alpha(v)beta(6) in vivo by positron emission tomography (PET) using the peptide [(18)F]FBA-A20FMDV2. Here, we describe improved alpha(v)beta(6) imaging agents and test their efficacy in a mouse model with endogenous alpha(v)beta(6) expression. The modified compounds maintained high affinity for alpha(v)beta(6) and >1,000-fold selectivity over related integrins (by ELISA) and showed significantly improved alpha(v)beta(6)-dependent binding in cell-based assays (>60% binding versus <10% for [(18)F]FBA-A20FMDV2). In vivo studies using either a melanoma cell line (transduced alpha(v)beta(6) expression) or the BxPC-3 human pancreatic carcinoma cell line (endogenous alpha(v)beta(6) expression) revealed that the modified compounds showed significantly improved tumor retention. This, along with good clearance of nonspecifically bound activity, particularly for the new radiotracer [(18)F]FBA-PEG(28)-A20FMDV2, resulted in improved PET imaging. Tumor/pancreas and tumor/blood biodistribution ratios of >23:1 and >47:1, respectively, were achieved at 4 hours. Significantly, [(18)F]FBA-PEG(28)-A20FMDV2 was superior to 2-[(18)F]fluoro-2-deoxy-d-glucose ([(18)F]FDG) in imaging the BxPC-3 tumors. Pancreatic ductal adenocarcinoma is highly metastatic and current preoperative evaluation of resectability using noninvasive imaging has limited success, with most patients having metastases at time of surgery. The fact that these tumors express alpha(v)beta(6) suggests that this probe has significant potential for the in vivo detection of this malignancy, thus having important implications for patient care and therapy.

Basal cell carcinoma (BCC) is the most prevalent cancer in the Western world and its incidence is increasing. The pathogenesis of BCC involves deregulated Sonic hedgehog signaling, leading to activation of the Gli transcription factors. Most BCCs have a nodular growth pattern, and are indolent, slow-growing, and considered "low-risk" lesions. In contrast, the "high-risk" morphoeic variant, which causes significant morbidity, has an infiltrative growth pattern, and is so-called because of its densely fibrous stroma. As alpha v beta 6 is capable of promoting both carcinoma invasion and fibrosis, we examined the expression of this integrin in BCCs and found that the morphoeic type showed significantly higher alpha v beta 6 expression than the nodular type (P = 0.0009). In order to examine the function of alpha v beta 6, we transfected the transcription factors Gli1 or Gli2 into NTERT, human keratinocytes to generate a BCC model. These cells expressed alpha v beta 6 and were invasive, although inhibition of alpha v beta 6 had no direct effect on cell invasion. However, the cells showed alpha v beta 6-dependent activation of transforming growth factor-beta1, which induced transdifferentiation of human fibroblasts into myofibroblasts. Paracrine secretion of hepatocyte growth factor/scatter factor by these myofibroblasts promoted c-Met-dependent tumor invasion in both Transwell and three-dimensional organotypic assays. These experimental in vitro findings were confirmed using human clinical samples in which we showed that the stroma of morphoeic BCC is myofibroblast-rich compared with nodular BCC (P = 0.0036), that myofibroblasts express hepatocyte growth factor/scatter factor, and that morphoeic BCCs are strongly c-Met-positive. These data suggest that alpha v beta 6-dependent transforming growth factor-beta1 activation induces both the infiltrative growth pattern and fibrotic stroma so characteristic of morphoeic BCC.

Expression of the epithelial-specific integrin alphavbeta6 is low or undetectable in most adult tissues but may be increased during wound healing and inflammation and is up-regulated dramatically by many different carcinomas, making alphavbeta6 a promising target for the in vivo detection of cancer using noninvasive imaging. In addition, alphavbeta6 is recognized as promoting invasion and correlates with aggressive behavior of human cancers and thus agents that recognize alphavbeta6 specifically in vivo will be an essential tool for the future management of alphavbeta6-positive cancers. Recently, we identified the peptide NAVPNLRGDLQVLAQKVART (A20FMDV2), derived from foot-and-mouth disease virus, as a potent inhibitor of alphavbeta6. Using flow cytometry and ELISA, we show that this peptide is highly selective, inhibiting alphavbeta6-ligand binding with a IC50 of 3 nmol/L, an activity 1,000-fold more selective for alphavbeta6 than for other RGD-directed integrins (alphavbeta3, alphavbeta5, and alpha5beta1). A20FMDV2 was radiolabeled on solid-phase using 4-[18F]fluorobenzoic acid, injected into mice bearing both alphavbeta6-negative and alphavbeta6-positive (DX3puro/DX3purobeta6 cell lines) xenografts and imaged using a small animal positron emission tomography (PET) scanner. Rapid uptake (<30 min) and selective retention (>5 h) of radioactivity in the alphavbeta6-positive versus the alphavbeta6-negative tumor, together with fast renal elimination of nonspecifically bound activity, resulted in specific imaging of the alphavbeta6-positive neoplasm. These data suggest that PET imaging of alphavbeta6-positive tumors is feasible and will provide an important new tool for early detection and improved management of many types of cancers.

Enhanced expression levels of integrin alphavbeta6 have been linked to more aggressive invasive carcinoma cell behavior and poorer clinical prognosis. However, how alphavbeta6 determines invasion and the dynamics of integrin alphavbeta6 regulation in tumor cells are poorly understood. We have identified the 35-kDa HS1-associated protein X-1 (HAX-1) protein as a novel binding partner of the beta6 cytoplasmic tail using a yeast two-hybrid screen. We show that alphavbeta6-dependent migration is blocked following small interfering RNA (siRNA)-mediated depletion of HAX-1 in oral squamous cell carcinoma cell lines. Using both siRNA and membrane-permeable peptides, we show that alphavbeta6-dependent migration and invasion require HAX-1 to bind directly to beta6 and thereby regulate clathrin-mediated endocytosis of alphavbeta6 integrins. Progression of oral cancer is associated with enhanced expression of alphavbeta6 and HAX-1 proteins in patient tissue. This report establishes that integrin endocytosis is required for alphavbeta6-dependent carcinoma cell motility and invasion and suggests that this process is an important mechanism in cancer progression.

Worldwide oral squamous cell carcinoma (OSCC) represents about 5.5% of all malignancies, with approximately 30,000 new cases each year in the United States. The integrin alpha(v)beta(6) and the enzyme cyclooxygenase-2 (COX-2) are implicated in OSCC progression and have been suggested as possible therapeutic targets. Each protein also is reported to identify dysplasias at high risk of malignant transformation, and current clinical trials are testing the efficacy of nonsteroidal anti-inflammatory drugs (NSAID) at preventing OSCC development. Given the probable increased expression of alpha(v)beta(6) and COX-2 in OSCC and the inhibition of several integrins by NSAIDs, we investigated whether NSAIDs affected alpha(v)beta(6)-dependent cell functions. We found that expression of both alpha(v)beta(6) and COX-2 was significantly higher in OSCC compared with oral epithelial dysplasias. Neither protein preferentially identified those dysplastic lesions that became malignant. Using OSCC cell lines, modified to express varying levels of alpha(v)beta(6), we assessed the effect of COX-2 inhibition on cell invasion. We found that the COX-2 inhibitor NS398 inhibited specifically alpha(v)beta(6)-dependent, but not alpha(v)beta(6)-independent, OSCC invasion in vitro and in vivo, and this effect was modulated through prostaglandin E(2) (PGE(2))-dependent activation of Rac-1. Transient expression of constitutively active Rac-1, or addition of the COX-2 metabolite PGE(2), prevented the anti-invasive effect of NS398. Conversely, RNA interference down-regulation of Rac-1 inhibited alpha(v)beta(6)-dependent invasion. These findings suggest that COX-2 and alpha(v)beta(6) interact in promoting OSCC invasion. This is a novel mechanism that, given the ubiquity of alpha(v)beta(6) expression by head and neck cancers, raises the possibility that NSAIDs could protect against OSCC invasion.

To identify genes involved in the melanocyte to malignant melanoma conversion, we have applied differential display to the comparison of syngeneic murine B16F10 (metastatic melanoma) and Melan-a-immortalized melanocyte cell lines. Approximately 7000 bands were analyzed, revealing approximately 80 to be differentially displayed. Reverse Northern blotting and subsequent Northern blotting confirmed the reproducible differential expression of four transcripts. Three B16F10-specific bands encode novel genes or partially sequenced cDNAs of unknown function. One Melan-a-specific band was found to be identical to the 3' end region of the mouse Annexin VI mRNA and shown to have a reduced message expression in B16F10 relative to Melan-a. Differential expression was confirmed at the protein level with Western blotting using a rabbit polyclonal antiserum. Immunohistochemistry of human melanoma specimens with this antiserum revealed a decrease or loss of Annexin VI expression as melanomas progressed from a benign to a more malignant phenotype. Our results provide further evidence for a potential role of Annexin VI in tumor suppression.

Human melanoma cell lines that express high constitutive levels of the metastasis-associated marker intercellular adhesion molecule 1 (ICAM-1) were found to secrete interleukin 1 (IL-1) in vitro. Experiments with neutralizing antibodies showed that this cytokine was responsible for their expression of ICAM-1 but not that of two other progression/metastasis markers, Muc-18 and Gp IIb/IIIa. The IL-1 present in melanoma-conditioned medium induced the expression of vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule 1, and ICAM-1 on human umbilical vein endothelial cells (ECs) in culture and increased the rate at which melanoma cells and ECs adhered to each other. IL-1-producing melanoma lines adhered significantly more rapidly to ECs than did non-IL-1-producing lines, and this enhancement was reduced by prior incubation of the melanoma cells with neutralizing anti-IL-1 antibodies. Similarly, endothelial cells treated with conditioned medium from IL-1-producing melanoma lines adhered significantly more rapidly to melanoma cells than did ECs treated with medium from non-IL-1-producing melanoma lines, and this enhancement was abolished by addition of anti-IL-1 antibodies to EC cultures in conditioned medium. Blocking antibodies to endothelial vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule 1, and ICAM-1 failed to inhibit melanoma-EC adhesion, but an antibody to tumor cell GpIIb/IIIa did block adhesion by up to 44%. The ability to secrete IL-1 could increase the metastatic potential of melanoma cells by stimulating tumor cell-EC adhesion.

Aluminum sulfonated phthalocyanine has potential as a suitable photosensitizer for use in the photodynamic therapy of cancer. In the present study, cellular uptake and retention of the individual mono-, di-, tri-, and tetrasulfonated derivatives (AlS1-4Pc) were examined in tissue culture and in normal and neoplastic tissue of tumor-bearing mice. Uptake and retention of the various derivatives by cells in tissue culture correlated inversely with the degree of sulfonation. Accordingly, Colo 26 cells in monolayer culture, 24 h after addition of 10 microM of appropriate photosensitizer, had accumulated approximately 25-fold more AlS1Pc than AlS3Pc and retained this species longer than more sulfonated derivatives. In contrast to these in vitro results, it was found that Colo 26 growing s.c. in BALB/c mice accumulated photosensitizer to a greater extent when the degree of sulfonation increased, such that A1S4Pc greater than AlS3Pc greater than AlS2Pc greater than AlS1Pc. By 24-48 h after the i.v. injection of 0.1 ml 2.27 mM solution of individual photosensitizer, the relative ratios of tumor:adjacent tissue varied from greater than 10:1 to greater than 2:1, showing that selective tumor uptake may be affected profoundly by the composition of the phthalocyanine compound. The livers and spleens of both normal and tumor-bearing mice, unlike other normal tissue, took up the sulfonated derivatives in an order that provided a mirror image of that observed in neoplastic tissue. These complex in vivo distribution and retention characteristics appear to be a consequence of relative hydrophilicity/hydrophobicity properties of the sulfonated species and indicate the extent to which these characteristics may influence photosensitizer distribution and accumulation.

The potency of chloroaluminum sulfonated phthalocyanine (ClAlSPc) as a photosensitizing agent for photodynamic therapy of cancer was evaluated in vivo by its ability to be taken up and retained by murine tumors of diverse histological origin. Antitumor effects following laser irradiation were evaluated by measurement of the tumor weights of dissected-out tumor masses. Three tumors (Colo 26, a colorectal carcinoma; M5076, a reticulum cell sarcoma; and UV-2237, a fibrosarcoma) growing s.c. in the flank region retained substantially greater quantities of ClAlSPc than did adjacent skin and muscle achieving peak values 24-48 h after the i.v. administration of ClAlSPc (10 mg/kg). The relative magnitude of ClAlSPc retention by these tumors was Colo 26 greater than M5076 greater than UV-2237. However, normal liver and spleen were organs which retained the greatest amounts of ClAlSPc even compared to the s.c. grown tumors and other normal tissues examined. Flow cytometric analysis of tumor cell suspensions obtained from collagenase-digested tumors showed that individual neoplastic cells were capable of taking up and retaining ClAlSPc. Photodynamic therapy, undertaken by i.v. administration of dye (5 mg/kg) followed 24 h later by local laser light irradiation (675 nm, 100 J), brought about significant (Colo 26, M5076, and 3LL tumors) and obvious but nonsignificant (UV-2237 tumor) reductions in tumor weights, as assessed 5 days later. Thus, selective tumor retention of ClAlSPc coupled with a significant response to red light produced dramatic alterations in cancer growth.