Inhibitor of differentiation-4 (Id4) stimulates pigmentation in melanoma leading to histiocyte infiltration.

TGF-ß and the inhibitors of differentiation (Id) are linked. Smad7 and other TGF-ß inhibitors can potently suppress melanomagenesis; however, little work examining Ids has been reported in melanoma, particularly for Id4. Here, we report that Id4, but not Id2 or Id3 expression, surprisingly, activated robust melanin production in xenografts of previously amelanotic (lacking pigment) 1205Lu/Smad7 (S7) cells. Fontana-Masson stain and de-novo expression of MART-1 and tyrosinase proteins confirmed melanin production. Additionally, pigment-laden CD163+ mouse histiocytes with areas of extensive necrosis were found throughout S7/Id4 tumors, but not in parental 1205Lu, S7/Id2 or S7Id3-derived tumors. Mechanistic investigation revealed increased nuclear M-microphthalmia-associated transcription factor (MITF) and MART-1 promoter activation following Id4 expression in 1205Lu and WM852 melanoma cells, suggesting broader implications for Id4 in melanin synthesis. In human tumors, melanin colocalized with Id4 expression establishing a correlation. Current chemotherapeutics for melanoma are only marginally effective. Immunotherapy provides the most promise, yet the role of innate immunity is poorly understood. Here, TGF-ß suppression followed by Id4 expression results in extensive melanin synthesis and robust histiocyte recruitment following tumorigenesis, a novel role for Id4. Our results suggest that TGF-ß suppression coupled with pigment overproduction triggers an innate immune response resulting in tumor necrosis.

Id2, Id3 and Id4 overcome a Smad7-mediated block in tumorigenesis, generating TGF-ß-independent melanoma.

The role for the inhibitors of differentiation (Ids) proteins in melanomagenesis has been poorly explored. In other cell types, Ids have been shown to contribute to cell proliferation, migration and angiogenesis and, along with a number of other genes, are direct downstream targets of the transforming growth factor (TGF)-ß pathway. Expression of Smad7, which suppress TGF-ß signaling, or synthetic TGF-ß inhibitors, was shown to potently suppress melanomagenesis. We found that endogenous Id2, Id3 and Id4 expression was elevated in 1205Lu versus 1205Lu cells constitutively expressing Smad7, indicating Ids may play a role in melanomagenesis. Therefore, the effects of Tet-inducible expression of Id2, Id3 or Id4 along with Smad7 in TGF-ß-dependent 1205Lu human melanoma cells were explored in vitro and in vivo. 1205Lu cells formed subcutaneous tumors in athymic mice, whereas cells expressing Smad7 failed to form tumors. However, 1205Lu cells expressing Smad7 along with doxycycline-induced Id2, Id3 or Id4 were able to overcome the potent tumorigenic block mediated by S7, to varying degrees. Conversely, Id small interfering RNA knockdown suppressed anchorage-independent growth of melanoma. Histology of tumors from 1205Lu cells expressing Smad7 + Id4 revealed an average of 31% necrosis, compared with 5.2% in tumors from 1205Lu with vector only. Downstream, Ids suppressed cyclin-dependent kinase inhibitors, and re-upregulated invasion and metastasis-related genes matrix metalloproteinase 2 (MMP2), MMP9, CXCR4 and osteopontin, shown previously to be downregulated in response to Smad7. This study shows that Id2, Id3 and Id4 are each able to overcome TGF-ß dependence, and establish a role for Ids as key mediators of TGF-ß melanomagenesis.

Sequestration of E12/E47 and suppression of p27KIP1 play a role in Id2-induced proliferation and tumorigenesis.

Id2 is a member of the helix-loop-helix (HLH) family of transcription regulators known to antagonize basic HLH transcription factors and proteins of the retinoblastoma tumor suppressor family and is implicated in the regulation of proliferation, differentiation, apoptosis and carcinogenesis. To investigate its proposed role in tumorigenesis, Id2 or deletion mutants were re-expressed in Id2(-/-) dermal fibroblasts. Ectopic expression of Id2 or mutants containing the central HLH domain increased S-phase cells, cell proliferation in low and normal serum and induced tumorigenesis when grafted or subcutaneously injected into athymic mice. Similar to their downregulation in human tumors, the expression of cyclin-dependent kinase inhibitors p27(KIP1) and p15(INK4b) was decreased by Id2; the former by downregulation of its promoter by the Id2 HLH domain-mediated sequestration of E12/E47. Re-expression of p27(KIP1) in Id2-overexpressing cells reverted the hyperproliferative and tumorigenic phenotype, implicating Id2 as an oncogene working through p27(KIP1). These results tie together the previously observed misregulation of Id2 with a novel mechanism for tumorigenesis.

UVB upregulates the bax promoter in immortalized human keratinocytes via ROS induction of Id3.

Id3 belongs to the inhibitor of differentiation family of helix-loop-helix transcription factors, important in proliferation, differentiation and apoptosis. We showed that Id3, but not Id2 or Id1, mediates the UVB-sensitization of immortalized keratinocytes by inducing caspase 9-dependent apoptosis. In this study, quantitative PCR analysis revealed a time-dependent increase in Id3 mRNA induced by UVB, dependent on reactive oxygen species. UVB upregulated promoter activity of Id3, but not Id2, at early time points, as shown by reporter assays and also stabilized Id3 mRNA, increasing its half-life from 10 to approximately 60 min. We next examined downstream events related to UVB-induced Id3 upregulation and investigated the effects of UVB or ectopic expression of Id3 on bax promoter activity. Regulatory elements in the bax promoter that mediate transcriptional activation by UVB and Id3, in the absence of p53, were identified. Bax promoter deletion analysis revealed that transcriptional activation by UVB involves a 738-bp region upstream from the transcription start site of bax. Mimicking the effects of UVB, ectopic expression of Id3 also upregulated bax mRNA and activated this 738-bp fragment. Mutational analysis of the transcription binding sites further showed that point mutations of the E-box region found in the 738-bp fragment, but not in a 174-bp fragment, completely abolished Id3- and UVB-inducible bax promoter activity, thus confirming the importance of Id3 and UVB-mediated Id3 upregulation in activating the bax promoter. These results suggest a mechanism whereby reactive oxygen species upregulation of Id3 relieves repression of bax via E-box-binding factors.

Increased expression of the E3 ubiquitin ligase RNF5 is associated with decreased survival in breast cancer.

The selective ubiquitination of proteins by ubiquitin E3 ligases plays an important regulatory role in control of cell differentiation, growth, and transformation and their dysregulation is often associated with pathologic outcomes, including tumorigenesis. RNF5 is an E3 ubiquitin ligase that has been implicated in motility and endoplasmic reticulum stress response. Here, we show that RNF5 expression is up-regulated in breast cancer tumors and related cell lines. Elevated expression of RNF5 was seen in breast cancer cell lines that became more sensitive to cytochalasin D- and paclitaxel-induced apoptosis following its knockdown with specific short interfering RNA. Inhibition of RNF5 expression markedly decreased cell proliferation and caused a reorganization of the actin cytoskeleton in response to stress in MCF-7 but not in p53 mutant breast cancer cells, suggesting a p53-dependent function. Significantly, high levels of RNF5 were associated with decreased survival in human breast cancer specimens. Similarly, RNF5 levels were higher in metastatic melanoma specimens and in melanoma, leukemia, ovarian, and renal tumor-derived cell lines, suggesting that increased RNF5 expression may be a common event during tumor progression. These results indicate that RNF5 is a novel regulator of breast cancer progression through its effect on actin cytoskeletal alterations, which also affect sensitivity of breast cancer cells to cytoskeletal targeting antineoplastic agents.

Expression of tumor necrosis factor--related apoptosis-inducing ligand receptors 1 and 2 in melanoma.

PURPOSE: The proapoptotic receptors tumor necrosis factor--related apoptosis-inducing ligand receptor 1 (TRAIL-R1) and TRAIL-R2 are targets of drugs in clinical development, and receptor expression levels may be important determinants of sensitivity to receptor agonists. We assessed TRAIL-R1 and TRAIL-R2 expression patterns in a large cohort of melanomas and benign nevi. EXPERIMENTAL DESIGN: We analyzed tissue microarrays containing 546 melanomas and 540 nevi using our automated quantitative method to measure protein levels in situ (AQUA). The system uses S100 to define pixels as melanoma (tumor mask) within the array spot and measures intensity of TRAIL-receptor expression using Cy5-conjugated antibodies within the mask. AQUA scores were correlated with clinical and pathologic variables. RESULTS: TRAIL-R1 and TRAIL-R2 expression was higher in melanomas than in nevi (P < 0.0001), and higher in primary than in metastatic specimens (P = 0.0031 and P < 0.0001, respectively). TRAIL-R1 and TRAIL-R2 expression exceeding the 95th percentile for nevi was found in 19% and 74% of melanoma specimens, respectively. Although on univariate analysis, high TRAIL-R2 expression correlated with increased survival (P = 0.0439), it was not associated with survival within the primary or metastatic subcohorts. TRAIL-R1 expression was not associated with survival. CONCLUSIONS: TRAIL-R1 and TRAIL-R2 expression is higher in malignant melanocytes than in their benign counterparts, suggesting that these receptors might be effective therapeutic targets in melanoma. Expression is higher in early-stage disease than in metastatic specimens, and expression exceeding that found in nevi is found in a substantially larger fraction of melanomas for TRAIL-R2 compared with TRAIL-R1. Assessment of baseline tumor TRAIL receptor expression may be important in analysis of clinical trials involving TRAIL receptor agonists.

Microfabricated blood vessels undergo neoangiogenesis.

The greatest ambition and promise of tissue engineering is to manufacture human organs. Before "made-to-measure" tissues can become a reality [1-3], however, three-dimensional tissues must be reconstructed and characterized. The current inability to manufacture operational vasculature has limited the growth of engineered tissues. Here, free-standing, small diameter blood vessels with organized cell layers that recapitulate normal biological functionality are fabricated using microfluidic technology. Over time in culture, the endothelial cells form a monolayer on the luminal wall and remodel the scaffold with human extracellular matrix proteins. After integration into three-dimensional gels containing fibroblasts, the microvessels sprout and generate extended hollow branches that anastomose with neighboring capillaries to form a network. Both the microfabricated vessels and the extended sprouts support perfusion of fluids and particles. The ability to create cellularized microvessels that can be designed with a diameter of choice, produced by the meter, and undergo angiogenesis and anastomoses will be an extremely valuable tool for vascularization of engineered tissues. To summarize, ultraviolet (UV) photo-crosslinkable poly(ethylene glycol) and gelatin methacrylate polymers used in combination with sheath-flow microfluidics allow for the fabrication of small diameter blood vessels which undergo neoangiogenesis as well as other developmental processes associated with normal human blood vessel maturation. Once mature, these vessels can be embedded; perfused; cryogenically stored and respond to stimuli such as chemokines and shear stresses to mimic native human blood vessels. The applications range from tissue-on-chip systems for drug screening, characterization of normal and pathologic processes, and creation and characterization of engineered tissues for organ repair.

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function.

"Data characterizing microfabricated human blood vessels created via hydrodynamic focusing".

This data article provides further detailed information related to our research article titled "Microfabricated Blood Vessels Undergo Neovascularization" (DiVito et al., 2017) [1], in which we report fabrication of human blood vessels using hydrodynamic focusing (HDF). Hydrodynamic focusing with advection inducing chevrons were used in concert to encase one fluid stream within another, shaping the inner core fluid into 'bullseye-like" cross-sections that were preserved through click photochemistry producing streams of cellularized hollow 3-dimensional assemblies, such as human blood vessels (Daniele et al., 2015a, 2015b, 2014, 2016; Roberts et al., 2016) [2], [3], [4], [5], [6]. Applications for fabricated blood vessels span general tissue engineering to organ-on-chip technologies, with specific utility in in vitro drug delivery and pharmacodynamics studies. Here, we report data regarding the construction of blood vessels including cellular composition and cell positioning within the engineered vascular construct as well as functional aspects of the tissues.

Evaluating the expression and prognostic value of TRAIL-R1 and TRAIL-R2 in breast cancer.

PURPOSE: The cell surface receptors tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) and TRAIL-R2 transmit apoptotic signals, and agents that activate these receptors are in clinical development. We sought to determine the expression and prognostic value of TRAIL-R1 and TRAIL-R2 in early-stage breast cancer. EXPERIMENTAL DESIGN: Tissue microarrays containing specimens from 655 breast cancer patients with 20-year follow-up were employed and evaluated with our automated quantitative analysis (AQUA) system. The system uses cytokeratin to define pixels as breast cancer (tumor mask) within the array spot, and measures intensity of TRAIL receptor expression using Cy5 conjugated antibodies within the mask. AQUA scores were correlated with clinical and pathologic variables. TRAIL-R1 and TRAIL-R2 expression were similarly studied on 95 unmatched normal breast specimens. RESULTS: TRAIL-R1 expression was not associated with survival. High TRAIL-R2 expression strongly correlated with decreased survival (P = 0.0005). On multivariate analysis, high TRAIL-R2 expression remained an independent prognostic marker, as did nodal status and tumor size. High TRAIL-R2 expression correlated strongly with lymph node involvement (P = 0.0003). TRAIL-R2 expression was stronger in malignant specimens than in normal breast epithelium (P < 0.0001). CONCLUSIONS: High TRAIL-R2 expression was independently associated with decreased survival in breast cancer. The biological basis and the sensitivity of high TRAIL-R2 expressing cells to TRAIL agonists and/or chemotherapy are subject to further investigation. Evaluation of TRAIL-R2 expression in early-stage breast cancer may identify a subset of patients requiring more aggressive or pathway-targeted adjuvant treatment. Clinical trials involving TRAIL-R2 agonists should stratify patients based on TRAIL-R2 expression.

Automated quantitative analysis of HDM2 expression in malignant melanoma shows association with early-stage disease and improved outcome.

The incidence of cutaneous malignant melanoma continues to increase every year, and this disease remains the leading cause of skin cancer death in industrialized countries. Despite the aggressive nature of advanced melanoma, there are no standard biological assays in clinical usage that can predict metastasis. This may be due, in part, to the inadequacy of reproducible assessment of protein expression using traditional immunohistochemistry. We have previously described a novel method of quantitative assessment of protein expression (AQUA) with the continuity and accuracy of an ELISA assay but with maintenance of critical spatial information. Here, we modify this technology for the evaluation of protein expression in melanoma. Using a tissue microarray cohort of 405 melanoma lesions and 17 normal skin samples, we analyzed expression of HDM2, the human homologue of murine double minute 2 with automated quantitative analysis. We show that expression levels in the nucleus are significantly higher in primary melanomas than in metastatic lesions. Furthermore, high levels of expression are predictive of better outcome. This study demonstrates that quantitative assessment of protein expression is useful in melanoma to validate potential tissue biomarkers and suggests that human homologue of murine double minute 2 may be a valuable prognostic tool for management of malignant melanoma.

Automated quantitative analysis of tissue microarrays reveals an association between high Bcl-2 expression and improved outcome in melanoma.

The addition of B-cell lymphoma 2 (Bcl-2) antisense to dacarbazine in the treatment of metastatic melanoma demonstrates improved response rates and progression-free survival when compared with dacarbazine alone. Studies on small cohorts of melanoma patients have shown variability in Bcl-2 expression (60%-96% positive). We performed quantitative analysis of Bcl-2 expression in a large patient cohort to assess the association with outcome. Tissue microarrays containing intact melanoma specimens representing 402 patients (339 with associated survival data) were analyzed with our AQUA system for automated quantitative analysis. Automated, quantitative analysis uses S100 to define pixels as melanoma (tumor mask) within the array spot and measures intensity of Bcl-2 expression using a Cy5 conjugated antibody within the mask. A continuous index score is generated, which is directly proportional to the number of molecules per unit area. Scores were divided into quartiles and correlated with clinical variables. High Bcl-2 expression was associated with better outcome in the entire cohort and among metastatic specimens only (P = 0.004 and P = 0.015, respectively). Expression was higher in primary than in metastatic specimens (P < 0.0001). There was no association between Bcl-2 expression and Breslow depth or Clark level. The diverse results within the literature may be due to use of small cohorts or variability in staining technique. These results suggest studies are needed to evaluate the association between quantitative assessment of Bcl-2 expression and response to Bcl-2 targeting therapy toward the goal of improved response rates to these drugs.

Microvessel manifold for perfusion and media exchange in three-dimensional cell cultures.

Integrating a perfusable microvasculature system in vitro is a substantial challenge for "on-chip" tissue models. We have developed an inclusive on-chip platform that is capable of maintaining laminar flow through porous biosynthetic microvessels. The biomimetic microfluidic device is able to deliver and generate a steady perfusion of media containing small-molecule nutrients, drugs, and gases in three-dimensional cell cultures, while replicating flow-induced mechanical stimuli. Here, we characterize the diffusion of small molecules from the perfusate, across the microvessel wall, and into the matrix of a 3D cell culture.

Id3 induces an Elk-1-caspase-8-dependent apoptotic pathway in squamous carcinoma cells.

Inhibitor of differentiation/DNA-binding (Id) proteins are helix-loop-helix (HLH) transcription factors. The Id protein family (Id1-Id4) mediates tissue homeostasis by regulating cellular processes including differentiation, proliferation, and apoptosis. Ids typically function as dominant negative HLH proteins, which bind other HLH proteins and sequester them away from DNA promoter regions. Previously, we have found that Id3 induced apoptosis in immortalized human keratinocytes upon UVB exposure, consistent with its role as a tumor suppressor. To investigate the role of Id3 in malignant squamous cell carcinoma (SCC) cells (A431), a tetracycline-regulated inducible system was used to induce Id3 in cell culture and mouse xenograft models. We found that upon Id3 induction, there was a decrease in cell number under low serum conditions, as well as in soft agar. Microarray, RT-PCR, immunoblot, siRNA, and inhibitor studies revealed that Id3 induced expression of Elk-1, an E-twenty-six (ETS)-domain transcription factor, inducing procaspase-8 expression and activation. Id3 deletion mutants revealed that 80 C-terminal amino acids, including the HLH, are important for Id3-induced apoptosis. In a mouse xenograft model, Id3 induction decreased tumor size by 30%. Using immunofluorescent analysis, we determined that the tumor size decrease was also mediated through apoptosis. Furthermore, we show that Id3 synergizes with 5-FU and cisplatin therapies for nonmelanoma skin cancer cells. Our studies have shown a molecular mechanism by which Id3 induces apoptosis in SCC, and this information can potentially be used to develop new treatments for SCC patients.

Clonal dominance of CD133+ subset population as risk factor in tumor progression and disease recurrence of human cutaneous melanoma.

Chemotherapeutic refractoriness of advanced cutaneous melanoma may be linked with melanoma-initiating cells, also known as melanoma stem cells. This study aimed to determine relative risk of clonal dominance of the CD133+ phenotype in tissues from melanoma patients with different clinical outcomes that could be applied to early diagnosis, prognosis or disease monitoring. Significant overexpression of CD133 (p<0.02) was observed by immunohistochemical staining in tissues from patients with recurrent disease versus those without disease recurrence. Relative risk analysis between these two groups suggested that the patients with recurrence or metastatic lesion had a greater than 2-fold overexpression of CD133. In addition, immunodetectable CD133 corroborated with upregulation of CD133 RNA levels (14- to 30-fold) as assessed by quantitative real-time reverse transcription-PCR (qRT-PCR) comparison of melanoma cell lines derived from patients with poor clinical outcomes and short overall survival (<10 months), vs. those derived from patients with good clinical outcomes and longer overall survival (>24 months). Further, cells derived from patients, and MACS-sorted according to their CD133 status retained their CD133-positivity (>95%) or CD133-negativity (>95%) for more than 8 passages in culture. CD133+ cells could repopulate and form tumors (p<0.03) in athymic NCr-nu/nu mice within 8 weeks while no tumors were observed with CD133- phenotype (up to 200,000 cells). Taken together, the study demonstrates, for the first time, that there exists a clonal dominance of a CD133+ population within the hierarchy of cells in cutaneous tissues from patients that have undergone successive progressive stages of melanoma, from primary to metastatic lesions. CD133, thus, provides a predictive marker of disease as well as a potential therapeutic target of high-risk melanoma.

Smad7 restricts melanoma invasion by restoring N-cadherin expression and establishing heterotypic cell-cell interactions in vivo.

The list of transforming growth factor-beta (TGF-ß)-related proteins in non-canonical TGF-ß signaling is growing. Examples include receptor-Smads directing micro-RNA processing and inhibitory-Smads, e.g. Smad7, directing cell adhesion. Human skin grafts with fluorescently tagged melanoma cells revealed Smad7-expressing cells positioned themselves proximal to the dermal-epidermal junction and failed to form tumors, while control cells readily invaded and formed tumors within the dermis. Smad7 significantly inhibited ß-catenin T41/S45 phosphorylation associated with degradation and induced a 4.5-fold increase in full-length N-cadherin. Cell adhesion assays confirmed a strong interaction between Smad7-expressing cells and primary dermal fibroblasts mediated via N-cadherin, while control cells were incapable of such interaction. Immunofluorescent analysis of skin grafts indicated N-cadherin homotypic interaction at the surface of both Smad7 cells and primary dermal fibroblasts, in contrast to control melanoma cells. We propose that Smad7 suppresses ß-catenin degradation and promotes interaction with N-cadherin, stabilizing association with neighboring dermal fibroblasts, thus mitigating invasion.

Long-term preservation of antigenicity on tissue microarrays.

Tissue microarrays have facilitated the evaluation of large cohort studies; however, there is little data on the best method for preserving sections once they are cut. We assessed three methods of storing precut breast cancer microarray slides: paraffin coating and storage in a nitrogen desiccator, either alone or in combination. We tested the durability of three antigens, cytokeratin, estrogen receptor, and Ki-67 on microarrays stored under these conditions for 3 months at room temperature. Staining was assessed with both manual scoring using traditional brown stain (0-3+) as well as automated scoring using fluorescently stained sections. Staining intensity was compared to that obtained from freshly cut slides. Slides stored under ambient conditions (room temperature and air) for 3 months exhibited marked degradation of all target antigens, in some cases resulting in slides that were virtually unreadable. We found that combined paraffin coating and nitrogen storage resulted in the best preservation of antigenicity, with retention of 72-99% of the antigenicity of a freshly cut slide, depending upon the marker and detection system used. The use of either paraffin coating or nitrogen storage alone protected slides to a lesser degree.