Epithelial Antigen Research Articles

Six-transmembrane epithelial antigens of the prostate comprise a novel inflammatory nexus in patients with pustular skin disorders

Pustular skin disorders are a category of difficult-to-treat and potentially life-threatening conditions that involve the appearance of neutrophil-rich pustules. The molecular basis of most pustular skin conditions has remained unknown. We sought to investigate the molecular basis of 3 pustular skin disorders: generalized pustular psoriasis (GPP), palmoplantar pustulosis (PPP), and acute generalized exanthematous pustulosis (AGEP). Microarray analyses were performed to profile genome-wide gene expression of skin biopsy specimens obtained from patients with GPP, PPP, or AGEP and healthy control subjects. Functional enrichment, gene network, and k-means clustering analyses were used to identify molecular pathways dysregulated in patients with these disorders. Immunohistochemistry and immunofluorescence were used to determine protein localization. Quantitative RT-PCR and ELISA were used to determine transcript and secreted cytokine levels. Small interfering RNA was used to decrease transcript levels. Molecules and pathways related to neutrophil chemotaxis emerged as common alterations in patients with GPP, PPP, and AGEP, which is consistent with the pustular phenotypes. Expression of two 6-transmembrane epithelial antigens of the prostate (STEAP) proteins, STEAP1 and STEAP4, was increased in patients' skin and colocalized with IL-36γ around neutrophilic pustules. STEAP1/4 expression clustered with and positively correlated with that of IL-1, the IL-36 family proteins, and CXCL1/8. STEAP4 expression was activated by cytokines and suppressed by inhibition of mitogen-activated protein kinase kinase 1/2, whereas STEAP1 expression appeared less prone to such dynamic regulation. Importantly, STEAP1/4 knockdown resulted in impaired induction of a broad spectrum of proinflammatory cytokines, including IL-1, IL-36, and the neutrophil chemotaxins CXCL1 and CXCL8. STEAP1/4 knockdown also reduced the ability of keratinocytes to induce neutrophil chemotaxis. Transcriptomic changes in 3 pustular skin disorders, GPP, PPP, and AGEP, converged on neutrophil chemotaxis and diapedesis and cytokines known to drive neutrophil-rich inflammatory processes, including IL-1 and members of the IL-36 family. STEAP1 and STEAP4 positively regulate the induction of proinflammatory neutrophil-activating cytokines.

Six-Transmembrane Epithelial Antigen of Prostate 1 (STEAP1) Has a Single b Heme and Is Capable of Reducing Metal Ion Complexes and Oxygen.

STEAP1, six-transmembrane epithelial antigen of prostate member 1, is strongly expressed in several types of cancer cells, particularly in prostate cancer, and inhibition of its expression reduces the rate of tumor cell proliferation. However, the physiological function of STEAP1 remains unknown. Here for the first time, we purified a mammalian (rabbit) STEAP1 at a milligram level, permitting its high-quality biochemical and biophysical characterizations. We found that STEAP1 likely assembles as a homotrimer and forms a heterotrimer when co-expressed with STEAP2. Each STEAP1 protomer binds one heme prosthetic group that is mainly low-spin with a pair of histidine axial ligands, with small portions of high-spin and P450-type heme. In its ferrous state, STEAP1 is capable of reducing transition metal ion complexes of Fe3+ and Cu2+. Ferrous STEAP1 also reacts readily with O2 through an outer sphere redox mechanism. Kinetics with all three substrates are biphasic with ∼80 and ∼20% for the fast and slow phases, respectively, in line with its heme heterogeneity. STEAP1 retained a low level of bound FAD during purification, and the binding equilibrium constant, KD, was ∼30 μM. These results highlight STEAP as a novel metal reductase and superoxide synthase and establish a solid basis for further research into understanding how STEAP1 activities may affect cancer progression.

Flow Cytometry of Nonhematopoietic Neoplasms

Many epithelial neoplasms can be analyzed by flow cytometry (FC), particularly from serous cavity effusion samples, using EpCAM, a cell adhesion molecule expressed on most normal epithelial cells and expressed at a higher level in most epithelial neoplasms. A simple 3-color flow cytometric panel can provide a high sensitivity and specificity compared to cytomorphology. FC provides more rapid immunophenotyping than conventional immunohistochemical staining, can identify rare malignant cells that could be missed by a cytological exam alone, and can be utilized to evaluate limited samples such as cerebrospinal fluid or fine-needle aspiration samples. Flow cytometric analysis for epithelial antigens can be combined with DNA ploidy analysis or assessment of the nucleus-to-cytoplasm ratio. Panels of flow cytometric markers are useful for the assessment of pediatric nonhematopoietic neoplasms, including neuroblastomas, primitive neuroectodermal tumors, Wilms' tumor, rhabdomyosarcomas, germ cell tumors, and hemangiopericytomas, as well as small-round-blue-cell tumors in adults, including small-cell carcinomas.

Sebaceoma: a clinicopathological analysis of 31 cases

Objective To analyze clinicopathologic features of sebaceoma. Methods Clinical, pathologic and immunohistochemical findings from 31 cases of sebaceoma were retrospectively analyzed. The clinicopathologic features of sebaceoma were investigated. Results There were 9 males and 22 females. The patients′ age was 53.90 ± 15.40 years, and the clinical course was 9.41 ± 13.75 years. Sebaceoma predominantly affected the face. The common lesion of sebaceoma was red, yellowish-red, skin-colored or slight brown papules, with no subjective symptoms in most cases. Histopathologically, neoplasms had symmetric structures, and were located in the dermis. Epidermal involvements were found in 9 cases. The neoplasm cells were mainly composed of basaloid cells, a few mature sebocytes and some transition cells. The proportion of mature sebocyts was less than 1% in 26 cases, less than 20% in 2 cases, and 20%-40% in 3 cases. Mitoses were occasionally found in 5 cases. One patient was complicated by eccrine poroma. Varying amounts of ducts were found in all the patients. Immunohistochemical staining showed that epithelial membrane antigen was expressed on ducts and mature sebocytes in all the patients, while epithelial antigen was undetected in any of the patients. Carcinoembryonic antigen, androgen receptor and D2-40 were found in 20, 24 and 28 patients with sebaceoma, respectively. Conclusions The diagnosis of sebaceoma mainly depends on histopathological examination. Combined immunohistochemical detection of epithelial membrane antigen, androgen receptor and D2-40 is beneficial to its differential diagnosis. Key words: Sebaceoma; Carcinoembryonic antigen; Receptors, androgen; Epithelial membrane antigen; Epithelial antigen; D2-40

Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions

To study the ability of human spermatogonial stem cells (hSSCs) to proliferate invitro under mouse spermatogonial stem cell (mSSC) culture conditions. Experimental basic science study. Reproductive biology laboratory. Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment. Testicular cells used to create invitro cell cultures corresponding to the following groups: [1] unsorted human testicular cells, [2] differentially plated human testicular cells, and [3] cells enriched with major histocompatibility complex class 1 (HLA-)/epithelial cell surface antigen (EPCAM+) in coculture with inactivated testicular feeders from the same patient. Analyses and characterization including immunocytochemistry and quantitative reverse-transcription polymerase chain reaction for somatic and germ cell markers, testosterone and inhibin B quantification, and TUNEL assay. Putative hSSCs appeared in singlets, doublets, or small groups of up to four cells invitro only when testicular cells were cultured in StemPro-34 medium supplemented with glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated cell sorting with HLA-/EPCAM+ resulted in an enrichment of 27% VASA+/UTF1+ hSSCs, compared to 13% in unsorted controls. Coculture of sorted cells with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm2 after 2weeks invitro compared with unsorted cells (61 hSSCs/cm2) and differentially plated cells (49 hSSCS/cm2). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4weeks invitro. We found that hSSCs show limited proliferation invitro under mSSC culture conditions. Coculture of HLA-/EPCAM+ sorted cells with testicular feeders improved the germ cell/somatic cell ratio.

Abstract 1677: CLCA2 interaction with EVA1 promotes mammary epithelial differentiation

Abstract CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized HMEC caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The TMS primary sequence of CLCA2 was found to be conserved throughout mammals. Moreover, competitive inhibition of the interaction by ectopic expression of the TMS caused partial EMT, suggesting that the interaction is important for differentiation. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines. Citation Format: Grace N.T. Ramena, Yufang Yin, Yang Yu, Vijay Walia, Randolph C. Elble. CLCA2 interaction with EVA1 promotes mammary epithelial differentiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1677.

Abstract 2794: Six-transmembrane epithelial antigen of prostate 4 (STEAP4): an important link between hypoxia, inflammation, nutrition and tumorigenesis in the intestine

Abstract Hypoxia occurred in the inflammatory foci can further increase the pro-inflammatory response. Moreover, inflammatory hypoxia is critical for altering tissue metabolism and nutritional response, which can further sustain the inflammation and drive tumorigenesis. However, the mechanisms that link hypoxia, nutrition, inflammation and tumorigenesis are not clear. Through genome-wide expression analysis we identified six-transmembrane epithelial antigen of prostate 4 (STEAP4) as a gene highly induced by hypoxia. STEAP4 is an oxidoreductase that regulates inflammatory stress, fatty acid, glucose and iron metabolism. Here we demonstrate that the increase in STEAP4 is dependent on epithelial HIF-2α in mouse models of colitis. Luciferase reporter assay and ChIP assay confirmed that STEAP4 is a HIF-2α target gene. Compared to normal healthy controls, the gene expression of STEAP4 was highly induced in tissues from colitis patients. Transgenic mice overexpressing GFP-tagged STEAP4 specifically in intestinal epithelial cells demonstrated that STEAP4 was expressed primarily on the plasma and mitochondrial membrane. Interestingly, these mice had no change in serum and tissue minerals except for a slight increase in intestinal iron, specifically mitochondrial iron, but no change in systemic iron levels. STEAP4 transgenic mice were highly susceptible to acute experimental colitis model. Moreover, STEAP4 was increased in human colorectal cancer (CRC) and predicts poor prognosis. Consistently, tumor numbers in the STEAP4 transgenic overexpression mice were increased compared to wild-type littermates in a colitis-associated CRC tumor model. Mechanistically, overexpression of STEAP4 increased oxidative stress and DNA damage in the intestine. In conclusion, we have identified a novel gene induced in colitis and CRC that could link hypoxia-induced metabolic changes, inflammation and cancer. Citation Format: Xiang Xue, Yatrik Shah. Six-transmembrane epithelial antigen of prostate 4 (STEAP4): an important link between hypoxia, inflammation, nutrition and tumorigenesis in the intestine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2794.

Abstract LB-253: Control of tumor growth in vivo by a synthetic multi-antigen DNA immune therapy for prostate cancer

Abstract Prostate cancer (PCa) is one of the leading causes of cancer deaths among men with limited treatment options. Accordingly, new approaches, such as immunotherapy, may represent important approaches for PCa treatment. Electroporation (EP) delivered DNA vaccines has recently shown promising results for therapeutic immunotherapy strategies has been limited to single antigen and epitope targets with limited success. We hypothesized that a broader collection of antigens adjuvanted by plasmid encoded IL-12 would bypass immune tolerance and improve the breadth and effectiveness of a PCa immunotherapy approach. We tested this hypothesis in NHP for immune tolerance effects as well as in the highly relevant TRAMP-C2 challenge model. We developed highly optimized DNA vectors encoding consensus antigens for important PCa targets prostate-specific antigen (SynCon PSA), prostate-specific membrane antigen (SynCon PSMA), and human six-transmembrane epithelial antigen of the prostate (STEAP). In mice the vaccines demonstrated potent IFNγ production by ELISpot (2740 SFU) and robust immune responses in the CD4+ (0.53%) and CD8+ (3.0%) T cell compartments. Further, sera from immunized mice reacted in ELISA with relevant targets and specifically stained LNCaP cells, a human PCa cell line, as well as human PCa tumor sections, supporting that the vaccine antigens induced relevant antibody responses. Vaccination of Rhesus Macaques, which share greater than 98% identity with humans, showed robust anti-PSA, PSMA and STEAP IFNγ production (612 SFU), potential for cytotoxic T cell function, and antigen specific seroconversion supporting the ability of these constructs to break tolerance. The therapeutic potential of PSMA, STEAP, and the combination of PSMA and STEAP, alone or with the molecular adjuvant IL-12, was evaluated in mice in the TRAMP-C2 tumor model. Alone, PSMA, STEAP or PSMA+STEAP demonstrated prolonged survival and a modest impact on tumor growth. However, the combination of synthetic vaccine antigens with IL-12 resulted in 100% efficacy in treatment and clearance of tumors resulting in 100% survival. These data support further study of this novel immune therapy of PCa. Citation Format: Bernadette Ferraro, Jewell N. Walters, Emma L. Reuschel, Amritha Balakrishnan, Matthew P. Morrow, Amir S. Khan, Niranjan Y. Sardesai, Laurent M. Humeau, David B. Weiner. Control of tumor growth in vivo by a synthetic multi-antigen DNA immune therapy for prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-253.

Transcriptome analysis of copper homeostasis genes reveals coordinated upregulation of SLC31A1,SCO1, and COX11 in colorectal cancer

Copper homeostasis and distribution is strictly regulated by a network of transporters and intracellular chaperones encoded by a group of genes collectively known as copper homeostasis genes (CHGs). In this work, analysis of The Cancer Genome Atlas database for somatic point mutations in colorectal cancer revealed that inactivating mutations are absent or extremely rare in CHGs. Using oligonucleotide microarrays, we found a strong increase in mRNA levels of the membrane copper transporter 1 protein [CTR1; encoded by the solute carrier family 31 member 1 gene (SLC31A1 gene)] in our series of colorectal carcinoma samples. CTR1 is the main copper influx transporter and changes in its expression are able to induce modifications of cellular copper accumulation. The increased SLC31A1 mRNA level is accompanied by a parallel increase in transcript levels for copper efflux pump ATP7A, copper metabolism Murr1 domain containing 1 (COMMD1), the cytochrome C oxidase assembly factors [synthesis of cytochrome c oxidase 1 (SCO1) and cytochrome c oxidase copper chaperone 11 (COX11)], the cupric reductase six transmembrane epithelial antigen of the prostate (STEAP3), and the metal‐regulatory transcription factors (MTF1, MTF2) and specificity protein 1 (SP1). The significant correlation between SLC31A1,SCO1, and COX11 mRNA levels suggests that this transcriptional upregulation might be part of a coordinated program of gene regulation. Transcript‐level upregulation of SLC31A1,SCO1, and COX11 was also confirmed by the analysis of different colon carcinoma cell lines (Caco‐2, HT116, HT29) and cancer cell lines of different tissue origin (MCF7, PC3). Finally, exon‐level expression analysis of SLC31A1 reveals differential expression of alternative transcripts in colorectal cancer and normal colonic mucosa.

Bufalin suppresses cancer stem-like cells in gemcitabine-resistant pancreatic cancer cells via Hedgehog signaling.

Cancer stem cells (CSCs) are important in cancer, as these cells possess enhanced tumor-forming capabilities and are resistant to current anticancer therapies. Agents with the ability to suppress CSCs are likely to provide novel opportunities for combating tumor proliferation and metastasis. The present study aimed to evaluate the effects of bufalin on pancreatic CSCs in vivo and in vitro. Using a serum-free suspension culture, tumor spheres were enriched in a gemcitabine-resistant human pancreatic cancer cell line, which had a higher percentage of CSCs, and western blotting, flow cytometry, and colony and tumor formation assays were used to demonstrate that these sphere cells exhibited CSC characteristics. Using these cancer stem-like cells as a model, the present study examined the effect of bufalin on pancreatic CSCs. It was demonstrated that bufalin inhibited the number of tumor spheres, and western blotting and immunohistochemical assays showed that the expression levels of CD24 and epithelial specific antigen (ESA) were downregulated by bufalin. Furthermore, in a subcutaneous xenograft model of implanted gemcitabine-resistant MiaPaCa2 cells, bufalin inhibited tumor growth and prolonged the duration of tumor formation. Additionally, the expression levels of CD24 and ESA were inhibited in the bufalin-treated mice. Notably, in another cancer model injected with tumor cells via the tail vein, fewer metastatic lesions were detected in the group in which tumor cells were pretreated with bufalin in vitro, compared with those without pretreatment. Of note, the Hedgehog (Hh) signaling pathway was found to be inhibited in the bufalin-treated cells. Taken together, these results suggested that bufalin suppressed pancreatic CSCs in gemcitabine-resistant MiaPaCa2 cells, and the Hh signaling pathway may be involved in this process.

Treatment of liver cancer in vitro and in mice by monoclonal antibody targeting epithelial specific antigen-positive liver cancer stem cells in combination with cisplatin

To investigate the biological characteristics of monoclonal antibodies against human liver cancer stem cells and its therapeutic effect in combination with cisplatin in the treatment of hepatocellular carcinoma. Cell culture in serum-free medium and PKH26 staining were used to determine the existence of cancer stem cells in human liver Bel7402-V3 cell line. The co-expression of antigen recognized by monoclonal antibody (McAb) 15D2 and epithelial specific antigen (ESA) and PKH26-positive cells in the Bel7402-V3 cells were detected by immunofluorescence assay. Serum-free suspension culture was used to detect the self-renewal ability of 15D2-positive Bel7402-V3 cells sorted by flow cytometry and the effect of 15D2 on the self-renewal ability of Bel7402-V3 cells. The effect of 15D2 on cisplatin resistance in the cells was examined by CCK8 method. The inhibitory effect of 15D2 combined with cisplatin on the transplanted tumor growth in mice was also observed. Single PKH26-positive cells were observed in the Bel7402-V3 cell spheroids cultured for 11 days. Immunofluorescence assay showed that the 15D2-recognized antigen could be conjugated with PKH26 and ESA and co-localized on Bel7402-V3 cells. The spheroid formation rate of 15D2-positive cells in serum-free medium was significantly higher than that of 15D2-negative cells [(30.4±3.4)% vs. (8.8±1.8)%, P<0.01]. The cisplatin resistance of 15D2-positive cells was obviously higher than that of 15D2-negative cells (IC50: 1.014 μmol/L vs. 0.365 μmol/L). McAb 15D2 significantly suppressed the spheroid formation of Bel7402-V3 cells, with an inhibition rate of 37.5%. McAb 15D2 also notably inhibited the cisplatin resistance of Bel7302-V3 cells. The IC50 was 0.211 μg/ml in the 15D2 group and 0.325 μg/ml in the control group. The mouse experiment showed that the tumor growth rates of 50 mg/kg, 25 mg/kg and 12.5 mg/kg 15D2-treatment groups were 82.6%, 71.4% and 60.0%, respectively; that of the 50 mg/kg 15D2 + cisplatin group was 91.0%, and that of the cisplatin monotherapy was 56.7%. McAb 15D2 is a functional monoclonal antibody targeting liver cancer stem cells, which could be a potential monoclonal antibody drug for the stem cell-targeted therapy of liver cancer.

Do pancreatic cancer (PDA) stem cell markers predict biologic behavior?

4112Background: Studies indicate heterogeneous clinical behavior arises in part from variant clones and cancer stem cells (CSC) which are CD44 (+) and Epithelial Specific Antigen (ESA) (+) (Lee, 20...

Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity

ABSTRACTPediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients' immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here, the specific recognition and lytic potential of transgenic allo-restricted CD8+ T cells, directed against the ES-associated antigen 6-transmembrane epithelial antigen of the prostate 1 (STEAP1), was examined. Following repetitive STEAP1130 peptide-driven stimulations with HLA-A*02:01+ dendritic cells (DC), allo-restricted HLA-A*02:01− CD8+ T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, T cell receptors' (TCR) α- and β-chains were identified, cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01− primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2−/−γc−/− mouse model. Initially generated transgenic T cells specifically recognized STEAP1130-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific interferon-γ (IFNγ) release, lysed cells and inhibited growth of HLA-A*02:01+ ES lines more effectively than HLA-A*02:01− ES lines. In vivo tumor growth was inhibited more effectively with transgenic STEAP1130-specific T cells than with unspecific T cells. Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1-expressing HLA-A*02:01+ ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1-expressing tumors.

The Prognostic Role of STEAP1 Expression Determined via Immunohistochemistry Staining in Predicting Prognosis of Primary Colorectal Cancer: A Survival Analysis.

STEAP1 (six transmembrane epithelial antigen of the prostate 1) is a transmembrane protein that functions as a potential channel or transporter protein. It is overexpressed in certain cancers and is viewed as a promising therapeutic target. However, the prognostic role of STEAP1 is still controversial, and no role for STEAP1 has yet been indicated in colorectal cancer. The aim of this study was to investigate the possible association of STEAP1 expression with colorectal cancer prognosis. STEAP1 expression was analyzed by immunohistochemical staining of a tissue array of 165 cancer specimens from primary colorectal cancer patients. The mean and medium follow-up times after surgery were 5.1 and 3.9 years, respectively. A total of 139 patients died during the 13 years of follow-up in the survey period. The prognostic value of STEAP1 with respect to overall survival was analyzed by Kaplan-Meier analysis and Cox proportional hazard models. In total, 164 samples displayed detectable STEAP1 expression in the cytoplasm and membrane. Low STEAP1 expression was correlated with poor overall survival (five-year survival: 33.7% vs. 57.0%, low expression vs. high expression, p = 0.020). Accordingly, multivariate analysis identified low STEAP1 expression as an independent risk factor (hazard ratio = 1.500, p = 0.018), especially in elderly patients or those with late stage cancers, late T values, and early N values. We suggest that analysis of STEAP1 expression by immunohistochemical staining could serve as an independent prognostic marker for colorectal patients. This finding should be validated by other investigative groups.

Immunogenicity and efficacy of the novel cancer vaccine based on simian adenovirus and MVA vectors alone and in combination with PD-1 mAb in a mouse model of prostate cancer.

Prostate cancer possesses several characteristics that make it a suitable candidate for immunotherapy; however, prostate cancer vaccines to date demonstrate modest efficacy and low immunogenicity. The goal of the present pre-clinical study was to explore the immunogenic properties and protective efficacy of a novel prostate cancer immunotherapy based on the heterologous prime–boost viral-vectored vaccination platform. The simian adenovirus, ChAdOx1, and modified vaccinia Ankara virus, MVA, encoding a prostate cancer-associated antigen, the six transmembrane epithelial antigen of the prostate 1 (STEAP1), induced strong sustained antigen-specific CD8+ T-cell responses in C57BL/6 and BALB/c male mice. Unexpectedly, the high vaccine immunogenicity translated into relatively low protective efficacy in the murine transplantable and spontaneous models of prostate cancer. A combination of the vaccine with PD-1 blocking antibody significantly improved survival of the animals, with 80 % of mice remaining tumour-free. These results indicate that the ChAdOx1–MVA vaccination regime targeting STEAP1 combined with PD-1 therapy might have high therapeutic potential in the clinic.

Sa1822 Epigenetic Control of Colonic Epithelial Antigen Processing, Barrier Function, and the Microbiome via Methyl-CpG Binding Domain Protein 2

Sa1822 Epigenetic Control of Colonic Epithelial Antigen Processing, Barrier Function, and the Microbiome via Methyl-CpG Binding Domain Protein 2

Commentary: Heart Failure with Preserved Ejection Fraction Induces Beiging in Adipose Tissue.

Commentary: Heart Failure with Preserved Ejection Fraction Induces Beiging in Adipose Tissue.

CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation.

CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized human mammary epithelial cells (HMEC) caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The primary sequence of the CLCA2 TMS was found to be conserved in CLCA2 orthologs throughout mammals, suggesting that its interaction with EVA1 co-evolved with the mammary gland. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines.

DOP004. Epigenetic control of colonic epithelial antigen processing, barrier function, and the microbiome via methyl-CpG binding domain protein 2

DOP004. Epigenetic control of colonic epithelial antigen processing, barrier function, and the microbiome via methyl-CpG binding domain protein 2

Ectopic expression of the chemokine CXCL17 in colon cancer cells.

Background:The novel chemokine CXCL17 acts as chemoattractant for monocytes, macrophages and dendritic cells. CXCL17 also has a role in angiogenesis of importance for tumour development.Methods:Expression of CXCL17, CXCL10, CXCL9 and CCL2 was assessed in primary colon cancer tumours, colon carcinoma cell lines and normal colon tissue at mRNA and protein levels by real-time qRT–PCR, immunohistochemistry, two-colour immunofluorescence and immunomorphometry.Results:CXCL17 mRNA was expressed at 8000 times higher levels in primary tumours than in normal colon (P<0.0001). CXCL17 protein was seen in 17.2% of cells in tumours as compared with 0.07% in normal colon (P=0.0002). CXCL10, CXCL9 and CCL2 mRNAs were elevated in tumours but did not reach the levels of CXCL17. CXCL17 and CCL2 mRNA levels were significantly correlated in tumours. Concordant with the mRNA results, CXCL10- and CXCL9-positive cells were detected in tumour tissue, but at significantly lower numbers than CXCL17. Two-colour immunofluorescence and single-colour staining of consecutive sections for CXCL17 and the epithelial cell markers carcinoembryonic antigen and BerEP4 demonstrated that colon carcinoma tumour cells indeed expressed CXCL17.Conclusions:CXCL17 is ectopically expressed in primary colon cancer tumours. As CXCL17 enhances angiogenesis and attracts immune cells, its expression could be informative for prognosis in colon cancer patients.