Phosphatase and Tensin Homolog (PTEN) Expression as a Surrogate Biomarker Correlated With the Depth of Invasion in Cutaneous Malignant Melanoma.

Objective The aim of this study is to evaluate the expression of the phosphatase and tensin homolog (PTEN), which is a tumor suppressor gene that is implicated in the pathogenesis of cutaneous malignant melanoma, in normal skin and melanoma tissue samples. The study also aimed to correlate PTEN expression levels with various clinicopathological parameters of melanoma lesions, thus highlighting the utility of PTEN expression as a prognostic biomarker for melanoma. Study design Immunohistochemistry (IHC) staining was performed on tissue microarray samples representing normal skin and melanoma biopsies of different clinicopathological parameters. Tissue photomicrographs were evaluated with Aperio ImageScope, which has a positive-pixel-counting algorithm built in. Subsequently, a histochemical score (H-score) was derived from the percentage of positive cells (%-staining) and their staining intensity. The H-scores were averaged in groups of tissue samples representing the different melanomas' tumor (T), node (N), and distant metastasis (M), also known as TNM parameters, as set forth by the American Joint Committee on Cancer (AJCC) classification. The mean H-scores were statistically compared using a two-tailed unpaired t-test. Results The PTEN protein expression was measured by IHC and found to be correlated with tumor thickness (T), which is a reliable indicator for survival rates. Specifically, PTEN was significantly downregulated in tumors with a thickness over 2 mm (T3+T4) compared to tumors with a thickness at or below 2 mm (T1+T2). Conclusions The PTEN protein expression, as measured by immunohistochemistry, helped differentiate between tumors with a thickness over 2 mm and tumors with a thickness at or below 2 mm, suggesting PTEN as a potential surrogate marker for the melanoma's invasion depth along with possible prognostic implications. Longitudinal studies evaluating risk stratification based on the expression of PTEN are needed to establish the utility of this promising biomarker in the clinic as an adjunct for pathological examination.

DJ-1 Oncogene as a Potential Diagnostic and Prognostic Biomarker for Head and Neck Cancer.

Background Current methods used to diagnose and prognosticate oropharyngeal cancer have contributed to unfavorable patient survival rates that have not significantly improved for the last several decades. Precision medicine oncology relies on molecular diagnostics and biomarkers to supplement existing methods of detecting and prognosticating cancers. This study evaluated the expression of DJ-1, an oncogene that is implicated in the pathogenesis of oral squamous cell carcinoma (OSCC), the most common type of head and neck cancer, to determine its utility as a diagnostic and prognostic biomarker. Methodology Immunohistochemistry (IHC) was performed on 13 normal oral mucosa tissue samples and 143 OSCC tissue samples of varying histopathological grades. Computer-assisted image analysis was performed using the Aperio ImageScope software from Leica Biosystems (Buffalo Grove, IL), which utilizes an algorithm of positive pixel counting for the quantification of immunoreactivity and the percentage of positive cell staining, generating a histo-score (H-score). The comparisons of the average H-scores of the different groups were made using a two-tailed T-test with P ≤ 0.05 set as the level of significance. Results The study found a significant increase in DJ-1 expression in oral squamous cell carcinoma tissue samples in comparison to the normal oral mucosa tissue samples. Additionally, the study documented a significant upregulation in DJ-1 expression in the OSCC tissue samples with high histopathological grades compared to the OSCC tissue samples with low histopathological grades. Conclusions DJ-1 expression patterns were able to reliably differentiate between oral squamous cell carcinoma and the normal counterpart tissues of the oral mucosa, thereby highlighting its role as a potential diagnostic biomarker. Moreover, DJ-1 expression significantly correlates with the OSCC histological grade, which serves as an indicator of the differentiation status and a predictor of the biological behavior of malignant neoplasms, adding to DJ-1's potential utility as a prognostic biomarker for this common type of head and neck cancer.

Characterization of DJ-1, PTEN, and p-Akt as Prognostic Biomarkers in the Progression of Oral Squamous Cell Carcinoma.

Objective Oral cancer has a five-year survival rate of 68%, and the methods used to assess it still rely heavily on morphology. Protein biomarkers can potentially increase the predictive power of histopathological evaluation. This study aims to examine the expression of three closely linked proteins implicated in the pathogenesis of oral squamous cell carcinoma (OSCC); protein deglycase (DJ-1), an oncogene, phosphatase, and tensin homolog (PTEN), a tumor suppressor gene, and phosphorylated protein kinase B (p-Akt), the activated form of a vital serine/threonine kinase, which is involved in the oncogenesis of several human malignancies, throughout the tumor progression steps to establish their potential as prognostic biomarkers. Study design Western blot analysis was carried out using four different cell lines representing the successive steps of OSCC progression, including normal oral keratinocytes, dysplastic oral keratinocytes, locally invasive OSCC, and metastatic OSCC.  Results DJ-1 expression was found to be upregulated gradually throughout the successive steps of OSCC progression from normal to dysplastic to locally invasive to metastatic OSCC. PTEN expression showed an overall opposite trend. Interestingly, a significant downregulation of p-Akt was seen in the locally invasive OSCC cells, although it was followed by a significant increase in p-Akt expression in the metastatic OSCC cell line, which is consistent with the role of p-Akt in the motility and migration of cancer cells. Conclusion This study documented trends in expression patterns of three important signaling molecules, DJ-1, PTEN, and p-Akt, in normal, premalignant, and malignant oral keratinocytes. The oncogenic DJ-1 and tumor suppressor PTEN were expressed in a manner consistent with their respective roles in tumorigenesis, while p-Akt only showed a significant upregulation in the metastatic OSCC cells. Overall, all three proteins exhibited unique trends throughout the progressive stages of OSCC tumor progression, thereby adding to their potential utility as prognostic biomarkers for oral cancer patients.

Cornulin as a Potential Novel Biomarker for Cutaneous Squamous Cell Carcinoma.

Background This study aimed to evaluate the expression of an epidermal differentiation marker, cornulin, in cutaneous squamous cell carcinoma (cSCC). Cornulin has been found to be downregulated in various squamous cell carcinomas of other tissues; however, its expression in cSCC has never been studied. We predicted that cornulin expression in cSCC is reduced compared to the normal epidermis. Moreover, we hypothesized that an inverse relationship exists between cornulin expression and the loss of differentiation, as defined by histopathological grading of cSCC lesions. Methodology Samples of normal skin and cSCC lesions of variable histopathological grades were stained using immunohistochemistry. High-resolution tissue images were analyzed with Aperio ImageScope (Leica Biosystems) utilizing a positive-pixel-counting algorithm to quantify the staining intensity. Histo-score (H-score) was calculated based on staining intensity and percentage of positive cell staining. Mean H-scores were compared using an unpaired t-test. Results We documented cornulin expression in cSCC for the first time. Cornulin levels were downregulated by more than two-fold in cSCC compared to the normal epidermis. Additionally, we observed a 4.5-fold downregulation in cornulin expression in tumors with high histopathological grades when compared to low histopathological grade tumors. Conclusions Cornulin expression levels measured through immunohistochemistry staining can help distinguish among the different histopathological grades of cSCC. Therefore, we propose that cornulin detection can be an adjunct to pathological examination to evaluate the differentiation status of cSCC specimens. Longitudinal studies are needed to establish the utility of cornulin as a diagnostic and prognostic biomarker for cSCC.

Cornulin as a Prognosticator for Lymph Node Involvement in Cutaneous Squamous Cell Carcinoma.

Background Cornulin is an epidermal differentiation marker and a stress-related protein. Its expression patterns are likely to reflect the multi-step tumorigenesis process of cSCC, given its role as a tumor suppressor. The aim of this study is to evaluate the utility of Cornulin as a prognosticator for cutaneous squamous cell carcinoma (cSCC). Specifically, the correlation between Cornulin expression and the clinicopathological parameter of lymph node involvement (nodal status), which plays a major role in determining cSCC prognosis and recurrence. We predicted that Cornulin expression declines as cSCC tumors metastasize to regional lymph nodes. Methodology Tissue samples of cSCC lesions of variable nodal involvement status were stained using immunohistochemistry, and high-resolution images were acquired. Aperio ImageScope software (Leica Biosystems) equipped with a positive-pixel-counting algorithm was used to quantify the staining intensity. Subsequently, Cornulin immunoreactivity was calculated as a Histo-score (H-score) value, which is based on the staining intensity and the percentage of positively stained cells. Mean H-scores were compared between groups using an unpaired t-test. Results A significant inverse correlation was found between Cornulin expression levels and metastasis to the lymph nodes. Specifically, primary tumors with metastasis to regional lymph nodes (N1) exhibited 9.5-fold decrease in Cornulin immunoreactivity compared to the primary tumor samples without lymph node involvement (N0). Conclusion Cornulin was found to be significantly downregulated in primary tumors with lymph node metastases. Detection assays to measure Cornulin expression in cSCC primary tumors might aid in determining the nodal status in these patients and possibly help determine cases of occult lymph node metastasis or micrometastasis. Future clinical studies are needed to help establish Cornulin's role in enhancing the predictive power of histopathological examination and improving survival rates for patients suffering from this type of skin cancer.

Characterization of Cornulin as a Molecular Biomarker for the Progression of Oral Squamous Cell Carcinoma.

Introduction It has been shown that the expression of the epidermal differentiation marker, Cornulin, declines with the progression of squamous cell carcinomas of several tissue types. Objectives This study aims to examine Cornulin expression at the cellular level in various cell lines representative of the successive progression steps of oral squamous cell carcinoma (OSCC), a major type of head and neck cancer. This can pave the way for the utilization of this novel biomarker as a diagnostic and prognostic indicator for oral cancer and help guide treatment options. Study design Western blotting was performed to measure Cornulin expression levels using standardized cell lysates from four different cell lines representing the successive steps of OSCC progression. Specifically, primary gingival keratinocytes, dysplastic oral keratinocytes (DOK), squamous cell carcinoma 25 (SCC25) cells, and Detroit 562 cells were used to represent normal oral keratinocytes, DOKs, locally invasive OSCC cells, and metastatic OSCC cells, respectively. Results Cornulin expression was found to be downregulated with the progression from normal to premalignant to malignant cells. Quantitative analysis revealed that Cornulin is significantly downregulated by 3.4 folds in DOK cells, by 23.7 folds in SCC25 cells, and by 5.2 folds in Detroit 562 cells compared to normal gingival keratinocytes. Interestingly, Cornulin was upregulated by 4.5 folds in the metastatic Detroit 562 cell line compared to the locally invasive SCC25 cells. Conclusion Altogether, Cornulin proved to be differentially expressed at the cellular level in cell lines representative of the successive steps of OSCC progression. Specifically, we documented a gradual decrease in Cornulin expression with the progression from normal to premalignant to malignant cells. Notably, there is a significant increase in the expression of Cornulin in the metastatic cell line Detroit 562 compared to the locally invasive cell line SCC25, suggesting a possible correlation with the biological behavior and unique characteristics of these two different phenotypes.

Cripto-1 as a Key Factor in Tumor Progression, Epithelial to Mesenchymal Transition and Cancer Stem Cells.

Cripto-1 is an essential protein for human development that plays a key role in the early phase of gastrulation in the differentiation of an embryo as well as assists with wound healing processes. Importantly, Cripto-1 induces epithelial to mesenchymal transition to turn fixed epithelial cells into a more mobile mesenchymal phenotype through the downregulation of epithelial adhesion molecules such as E-cadherin, occludins, and claudins, and the upregulation of mesenchymal, mobile proteins, such as N-cadherin, Snail, and Slug. Consequently, Cripto-1's role in inducing EMT to promote cell motility is beneficial in embryogenesis, but detrimental in the formation, progression and metastasis of malignant tumors. Indeed, Cripto-1 is found to be upregulated in most cancers, such as breast, lung, gastrointestinal, hepatic, renal, cervical, ovarian, prostate, and skin cancers. Through its role in EMT, Cripto-1 can remodel cancer cells to enable them to travel through the extracellular matrix as well as blood and lymphatic vessels to metastasize to different organs. Additionally, Cripto-1 promotes the survival of cancer stem cells, which can lead to relapse in cancer patients.

The role of Nodal and Cripto-1 in human oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a common epithelial malignancy of the oral cavity. Nodal and Cripto-1 (CR-1) are important developmental morphogens expressed in several adult cancers and are associated with disease progression. Whether Nodal and CR-1 are simultaneously expressed in the same tumor and how this affects cancer biology are unclear. We investigate the expression and potential role of both Nodal and CR-1 in human OSCC. Immunohistochemistry results show that Nodal and CR-1 are both expressed in the same human OSCC sample and that intensity of Nodal staining is correlated with advanced-stage disease. However, this was not observed with CR-1 staining. Western blot analysis of lysates from two human OSCC line experiments shows expression of CR-1 and Nodal, and their respective signaling molecules, Src and ERK1/2. Treatment of SCC25 and SCC15 cells with both Nodal and CR-1 inhibitors simultaneously resulted in reduced cell viability and reduced levels of P-Src and P-ERK1/2. Further investigation showed that the combination treatment with both Nodal and CR-1 inhibitors was capable of reducing invasiveness of SCC25 cells. Our results show a possible role for Nodal/CR-1 function during progression of human OSCC and that targeting both proteins simultaneously may have therapeutic potential.

CMV-independent lysis of glioblastoma by ex vivo expanded/activated Vδ1+ γδ T cells.

Vδ2(neg) γδ T cells, of which Vδ1+ γδ T cells are by far the largest subset, are important effectors against CMV infection. Malignant gliomas often contain CMV genetic material and proteins, and evidence exists that CMV infection may be associated with initiation and/or progression of glioblastoma multiforme (GBM). We sought to determine if Vδ1+ γδ T cells were cytotoxic to GBM and the extent to which their cytotoxicity was CMV dependent. We examined the cytotoxic effect of ex vivo expanded/activated Vδ1+ γδ T cells from healthy CMV seropositive and CMV seronegative donors on unmanipulated and CMV-infected established GBM cell lines and cell lines developed from short- term culture of primary tumors. Expanded/activated Vδ1+ T cells killed CMV-negative U251, U87, and U373 GBM cell lines and two primary tumor explants regardless of the serologic status of the donor. Experimental CMV infection did not increase Vδ1+ T cell--mediated cytotoxicity and in some cases the cell lines were more resistant to lysis when infected with CMV. Flow cytometry analysis of CMV-infected cell lines revealed down-regulation of the NKG2D ligands ULBP-2, and ULBP-3 as well as MICA/B in CMV-infected cells. These studies show that ex vivo expanded/activated Vδ1+ γδ T cells readily recognize and kill established GBM cell lines and primary tumor-derived GBM cells regardless of whether CMV infection is present, however, CMV may enhance the resistance GBM cell lines to innate recognition possibly contributing to the poor immunogenicity of GBM.

Preclinical evaluation of ex vivo expanded/activated γδ T cells for immunotherapy of glioblastoma multiforme.

We have previously shown that expanded/activated γδ T cells from healthy donors are cytotoxic to GBM cell lines and primary GBM explants. In this report, we examined the therapeutic effect of intracranial infusion of expanded/activated γδ T cells on human minimal and established U251 tumor xenografts in athymic nude mice. Immunohistochemistry was used to determine the presence of NKG2D ligands on cell lines and tumors, and blocking studies were used to determine the effect of these ligands on γδ T cell recognition. Expanded/activated γδ T cells were prepared by 18-day culture in RPMI, human serum (HS), anti-CD2, IL-12, IFN-γ, and OKT-3. Anti-GBM activity of the cell product was assessed using in vitro cytotoxicity assays against the GBM cell line U251MG in suspension and in adherent culture. Ex vivo expanded/activated γδ T cells were of the effector/memory phenotype, expressed Th1 cytokines, and effectively killed U251 cells in vitro. Xenografts were prepared using a U251 cell line following transfection with a firefly luciferase gene to monitor tumor progression. Mice treated with γδ T cells showed slower progression of both new and established GBM xenografts versus mice that received vehicle only as determined by photon emission over time. Median survival was improved in all γδ T cell treated groups between 32 and 50 days by Kaplan-Meier analysis. U251 cells expressed ULBP-2 and ULBP-3, although blocking of these reduced in vitro cytotoxicity of γδ T cells to U251MG by only 33 and 25%, respectively. These studies show that expanded/activated γδ T cells can mediate killing of new or established GBM xenografts, reduce tumor progression, and constitute a potentially effective novel immunotherapeutic strategy against GBM.

Early biosignature of oxidative stress in the retinal pigment epithelium.

The retinal pigment epithelium (RPE) is essential for retinoid recycling and phagocytosis of photoreceptors. Understanding of proteome changes that mediate oxidative stress-induced degeneration of RPE cells may provide further insight into the molecular mechanisms of retinal diseases. In the current study, comparative proteomics has been applied to investigate global changes of RPE proteins under oxidative stress. Proteomic techniques, including 2D SDS-PAGE, differential gel electrophoresis (DIGE), and tandem time-of-flight (TOF-TOF) mass spectrometry, were used to identify early protein markers of oxidative stress in the RPE. Two biological models of RPE cells revealed several differentially expressed proteins that are involved in key cellular processes such as energy metabolism, protein folding, redox homeostasis, cell differentiation, and retinoid metabolism. Our results provide a new perspective on early signaling molecules of redox imbalance in the RPE and putative therapeutic target proteins of RPE diseases caused by oxidative stress.

Prohibitin as an oxidative stress biomarker in the eye.

Identification of biomarker proteins in the retina and retinal pigment epithelium (RPE) under oxidative stress may imply new insights into signaling mechanisms of retinal degeneration at the molecular level. Proteomic data from an in vivo mice model in constant light and an in vitro oxidative stress model are compared to controls under normal conditions. Our proteomic study shows that prohibitin is involved in oxidative stress signaling in the retina and RPE. The identity of prohibitin in the retina and RPE was studied using 2D electrophoresis, immunohistochemistry, western blot, and mass spectrometry analysis. Comparison of expression levels with apoptotic markers as well as translocation between mitochondria and the nucleus imply that the regulation of prohibitin is an early signaling event in the RPE and retina under oxidative stress. Immunohistochemical analysis of murine aged and diabetic eyes further suggests that the regulation of prohibitin in the RPE/retina is related to aging- and diabetes-induced oxidative stress. Our proteomic approach implies that prohibitin in the RPE and the retina could be a new biomarker protein of oxidative stress in aging and diabetes.

Cleavage of the retinal pigment epithelium-specific protein RPE65 under oxidative stress.

The regeneration of the 11-cis-retinyl imine chromophore of rhodopsin during the visual cycle and mechanisms that control this process are central questions in the field of vision research. The retinal pigment epithelium (RPE)-specific protein RPE65 is centrally involved in the isomerization and hydrolysis of all-trans-retinyl esters. In this study, we investigated RPE65 cleavage and potential regulatory mechanisms under oxidative stress conditions. The D407 RPE cell cultures were exposed to H(2)O(2) (100-1000 microM). Changes in the levels of RPE65 and proteins related to apoptosis were investigated using gel electrophoresis and western blotting. Mass spectrometry was used to confirm the identity of RPE65. C57BL/6J (M450) and C3HeB/FeJ (L450) mice were used for in vivo experiments. We found that a novel 45kDa truncated fragment of the RPE65 protein, designated RPE45, appears in RPE cells upon light exposure or oxidative stress. RPE45 is generated in vitro by recombinant caspases via an ubiquitination-dependent mechanism. Collectively, our results indicate that oxidative stress during the visual cycle results in cleavage of RPE65.

Tumour secreted grp170 chaperones full-length protein substrates and induces an adaptive anti-tumour immune response in vivo.

PURPOSE: We employed a grp170-secreting tumour cell system to determine whether tumour cells engineered to secrete grp170 generate an antitumour-specific immune response. Further, we examine the possibility that secreted grp170 can bind to and co-transport out of tumour cells full-length tumour antigens that may play a role in the anti-tumour immune response. MATERIALS AND METHODS: Wild type Colon-26 and Colon-26 engineered to secrete grp170 were subcutaneously inoculated into BALB/c mice. Tumour growth was monitored, and variations in immunoregulatory mechanisms were evaluated using immunohistochemistry, lymphocyte depletion, ELISpot assays, and Western blot analysis. RESULTS: Immunisation of animals with grp170-secreting tumour cells results in rejection of the tumour by induction of antigen-specific, CD8-dependent immune responses. The secreted grp170 is able to deliver full-length tumour antigens to the tumour microenvironment, thus making them available for uptake by antigen presenting cells (APCs) to initiate tumour-specific immune responses. CONCLUSIONS: These data parallel our studies showing that hsp110 or grp170 are able to chaperone full-length proteins, and when complexed with protein antigens and used as vaccines, these complexes elicit immune responses in vivo against the protein antigens. This cell-based approach has the potential to be utilised as a tumour-specific vaccine in tumours of various histological origins.

Characterization of Molecular Markers Indicative of Cervical Cancer Progression.

Cervical cancer originates with human papillomavirus (HPV) infection and progresses via histologically-defined premalignant stages. Here we compare normal cervical epithelium and patient-matched high grade squamous intraepithelial lesions (HSIL) with cervical carcinoma tissue from the same patient population (n=10 per group). Specimens were analyzed by combined laser capture microdissection and 2D-DIGE. Significant expression changes were seen with 53 spots resulting in identification of 23 unique proteins at the molecular level. These include eight that uniquely distinguish normal epithelium and HSIL and four that uniquely distinguish HSIL and carcinoma. In addition, one protein, cornulin, distinguishes all three states. Other identified proteins included differentiation markers, oncogene DJ-1, serpins, stress and interferon-responsive proteins, detoxifying enzymes, and serum transporters. A literature review, performed for all identified proteins, allowed most changes to be assigned to one of three causes: direct or indirect HPV oncoprotein interactions, growth selection during latency, or interactions in the lesion microenvironment. Selected findings were confirmed by immunohistochemistry using either frozen sections from the same cohort or formalin fixed paraffin embedded samples from a tissue microarray. Novel markers described here have potential applications for increasing the predictive value of current screening methods.

Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes.

BACKGROUND: Infection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features. RESULTS: Six replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE). The median within-group coefficient of variation was 19-21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23%) of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2%) were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1); and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27). CONCLUSION: This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.

Characterization of a putative ovarian oncogene, elongation factor 1alpha, isolated by panning a synthetic phage display single-chain variable fragment library with cultured human ovarian cancer cells.

PURPOSE: In an effort to identify cell surface targets and single short-chain antibody (scFv) for ovarian cancer therapy, we used a phage display approach to isolate an antibody with high reactivity against ovarian cancer. EXPERIMENTAL DESIGN: A phage scFv library was subjected to panning against human SK-OV-3 ovarian cancer cells. A clone with high reactivity was selected and tested in immunoperoxidase staining on a panel of normal tissues and ovarian carcinoma. Using immunoprecipitation, a differentially expressed band was analyzed by mass spectrometry. The antigen subclass was characterized with reverse transcription-PCR on cDNA library of normal tissues, and 91 ovarian cancer specimens, and correlated with clinicohistopathologic characteristics. RESULTS: Ninety-six individual scFv clones were screened in ELISA following panning. scFv F7 revealed high reactivity with ovarian cancer cell lines and showed intense staining of 15 fresh ovarian cancer specimens and no staining of a panel of normal tissues. A 40-kDa protein was identified to be translation elongation factor 1alpha1 (EEF1A1; P < 0.05). The expression of EEF1A2, a highly homologous and functionally similar oncogene, was found to be restricted only to the normal tissues of the heart, brain, and skeletal muscle. Aberrant EEF1A2 mRNA expression was found in 21 of 91 (23%) of ovarian cancer specimens and significantly correlated with increased likelihood of recurrence (P = 0.021). CONCLUSIONS: scFv F7 may represent an ovarian cancer-specific antibody against translation EEF1A family of translational factors. We propose that EEF1A2 may be a useful target for therapy of human ovarian cancer.

Extracellular targeting of endoplasmic reticulum chaperone glucose-regulated protein 170 enhances tumor immunity to a poorly immunogenic melanoma.

We have demonstrated previously that immunization with tumor-derived endoplasmic reticulum (ER) chaperone glucose-regulated protein 170 (grp170) elicits potent antitumor immunity. In the present study, we determine the impact of extracellular targeting grp170 by molecular engineering on tumor immunogenicity and potential use of grp170-secreting tumor cells as a cancer vaccine. grp170 depleted of ER retention sequence "KNDEL," when secreted by B16 tumor cells, maintained its highly efficient chaperoning activities and was significantly superior to both hsp70 and gp96. The continued secretion of grp170 dramatically reduced the tumorigenicity of B16 tumor cells in vivo, although the modification did not alter its transformation phenotype and cell growth rate. C57BL/6 mice that rejected grp170-secreting B16 tumor cells (B16-sgrp170) developed a strong CTL response recognizing melanocyte differentiation Ag TRP2 and were resistant to subsequent tumor challenge. B16-sgrp170 cells also stimulated the production of proinflammatory cytokines by cocultured dendritic cells. Depletion studies in vivo indicate that NK cells play a primary role in elimination of viable B16-sgrp170 tumor cells inoculated into the animals, whereas both NK cells and CD8(+) T cells are required for a long-term protection against wild-type B16 tumor challenge. Both the secreted and endogenous grp170, when purified from the B16 tumor, exhibited potent tumor-protective activities. However, the B16-sgrp170 cell appears to be more effective than tumor-derived grp170. Thus, molecular engineering of tumor cell to release the largest ER chaperone grp170 is capable of eliciting innate as well as adaptive immune responses, which may provide an effective cell-based vaccination approach for cancer immunotherapy.

Proteomic analysis of mitochondria-to-nucleus retrograde response in human cancer.

All tumors examined to date contain mutations in mitochondrial DNA (mtDNA). In addition, depletion of mtDNA is reported in a variety of tumors. Mitochondrial dysfunction resulting from changes in mtDNA invokes mitochondria-to-nucleus retrograde response in human cells. To identify proteins involved in retrograde response and their potential role in tumorigenesis, we carried out a comparative proteomic analysis using a cell line in which the mitochondrial genome was completely depleted (rho(0) cells lacking all mtDNA-encoded protein subunits), a cybrid cell line in which mtDNA was restored, and the parental cell line. Our comparative proteomic approach revealed marked changes in the cellular proteome and led us to identify quantitative changes in expression of several proteins. We found that subunits of complex I and complex III, molecular chaperones, and a protein involved in cell cycle control were downregulated and Inosine 5'-monophosphate dehydrogenase type 2 (IMPDH2) involved in nucleotide biosynthesis was upregulated in rho(0) cells. Our findings demonstrate that the expression of proteins is restored to wild type level by transfer of wild type mitochondria to rho(0) cells, suggesting that these proteins play key roles in retrograde response. To determine a potential role for identified retrograde responsive proteins in tumorigenesis, we analyzed the expression of UQCRC1 gene (encoding ubiquinol cytochrome-c reductase core protein I) in breast and ovarian tumors. We found that (1) UQCRC1 was highly expressed in breast (74%) and ovarian tumors (34%) and (2) the expression positively correlated with cytochrome c-oxidase (COXII) encoded by mtDNA. Our study opens an avenue for identification of retrograde proteins as potential tumor suppressors or oncogenes involved in carcinogenesis.

Heat shock proteins HSP70 and GP96: structural insights.

Several heat shock proteins (HSPs) act as potent adjuvants for eliciting anti-tumor immunity. HSP-based tumor vaccine strategies have been highly successful in animal models and are undergoing testing in clinical trials. It is generally accepted that HSPs, functioning as chaperones for tumor antigens, elicit tumor-specific adaptive immune responses. HSPs also appear to induce innate immune responses in an antigen-independent fashion. Innate responses generated by HSPs may contribute to anti-tumor immunity. Immunologically active chaperones with anti-tumor activity are referred to as "immunochaperones". Here, we review the studies that address the role of structural domains or regions of the immunochaperones HSP70 and GP96 that may be involved in the induction of adaptive or innate immune responses.