Robert K. Bright

Induction of Tumorigenesis and Metastasis by the Murine Orthologue of Tumor Protein D52

Expression studies have consistently identified tumor protein D52 (TPD52) overexpression in tumor cells. Murine TPD52 (mD52) shares 86% identity with the human orthologue. To study a possible role for TPD52 in transformation, 3T3 fibroblasts were transfected with the full-length cDNA for mD52. Expression of mD52 was confirmed by reverse transcription-PCR (RT-PCR), real-time PCR, and Western blot analysis compared with 3T3 and vector-transfected 3T3 (3T3.V), and the resultant cell line was designated 3T3.mD52. At 4 weeks, 3T3.mD52 gained a 2-fold increase in growth rate, lost contact inhibition, and exhibited a marked phenotype change. Further characterization revealed an acquired ability for anchorage-independent cell growth. To determine whether 3T3.mD52 had become tumorigenic, naïve, healthy, immunocompetent syngeneic mice were inoculated subcutaneously with varying cell doses. Tumors measuring >1 cm2 were detected 60 days postinoculation with 3T3.mD52, and a 50% subcutaneous tumor incidence was obtained with as few as 5 × 105 3T3.mD52 cells. Remarkably, when lungs from 3T3.mD52 tumor-bearing mice were analyzed, numerous tumor nodules were observed, ranging from nodules less than 10 to nodules too numerous to count (inoculation with 1 × 105 and 5 × 106 cells, respectively). Further support for the metastatic capacity of 3T3.mD52 was the demonstration that transforming growth factor (TGF)-βR1 (receptor) expression decreased and TGF-β1 secretion increased in 3T3.mD52 compared with 3T3 controls. cDNA microarray analysis showed a gene expression pattern that further supported mD52-induced transformation and metastasis. Together, these data suggest that mD52 expression in 3T3 cells initiated cellular transformation, tumorigenesis, and progression to metastasis. (Mol Cancer Res 2007;5(2):133–44)

Sperm Protein 17 Is Expressed in Human Somatic Ciliated Epithelia

It was once believed that sperm protein 17 (Sp17) was expressed exclusively in the testis and that its sole function was to bind to the oocyte during fertilization. However, immunohistochemistry of the human respiratory airways and reproductive systems show that it is abundant in ciliated cells but not in human cells with stereocilia and microvilli. The high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP)-binding motif within this region, suggest that Sp17 plays a regulatory role in a PKA-independent AKAP complex in both male germinal and ciliated somatic cells.

TUMOR-ASSOCIATED ANTIGENS: FROM DISCOVERY TO IMMUNITY

There is a renewed enthusiasm for therapeutic vaccination as a viable treatment for patients with cancer. Early tumor vaccines were comprised of whole tumor cells, fragments of tumor cells, or protein lysate from tumor cells. Limited results with these approaches led investigators to begin developing the next generation of cancer vaccines based on defined tumor-associated antigens (TAAs). Defining and characterizing TAAs for human cancer, development of new approaches for identifying TAAs, and novel strategies to deliver the antigens as potent therapeutic vaccines have all been the focus of intense research in the past decade and will continue to be the focus for decades tocome. tumor antigens vaccines antigen discovery tumor immunity

Nonredundant functions for tumor protein D52-like proteins support specific targeting of TPD52

Purpose:Tumor protein D52 (TPD52 or D52) is frequently overexpressed in breast and other cancers and present at increased gene copy number. It is, however, unclear whether D52 amplification and overexpression target specific functional properties of the encoded protein. Experimental Design: The expression of D52-like genes and MAL2 was compared in breast tissues using quantitative reverse transcription-PCR. The functions of human D52 and D53 genes were then compared by stable expression in BALB/c 3T3 fibroblasts and transient gene knockdown in breast carcinoma cell lines. In situ D52 and MAL2 protein expression was analyzed in breast tissue samples using tissue microarray sections. Results: The D52 (8q21.13), D54 (20q13.33), and MAL2 (8q24.12) genes were significantly overexpressed in breast cancer tissue (n = 95) relative to normal breast (n = 7; P ≤ 0.005) unlike the D53 gene (6q22.31; P = 0.884). Subsequently, D52-expressing but not D53-expressing 3T3 cell lines showed increased proliferation and anchorage-independent growth capacity, and reduced D52 but not D53 expression in SK-BR-3 cells significantly increased apoptosis. High D52 but not MAL2 expression was significantly associated with reduced overall survival in breast carcinoma patients (log-rank test, P < 0.001; n = 357) and was an independent predictor of survival (hazard ratio, 2.274; 95% confidence interval, 1.228-4.210; P = 0.009; n = 328). Conclusion: D52 overexpression in cancer reflects specific targeting and may contribute to a more proliferative, aggressive tumor phenotype in breast cancer.

SV40 Tag-specific cytotoxic T lymphocytes generated from the peripheral blood of malignant pleural mesothelioma patients

Abstract. Malignant pleural mesothelioma (MPM) is an aggressive cancer, with survival of less than one year following diagnosis and treatment with current protocols. Recent studies have demonstrated the presence of the simian virus 40 (SV40)-like, large tumor antigen (Tag) in nearly 60% of MPMs. SV40 Tag is a viral-encoded tumor-specific antigen, and thus a potential target for the induction of anti-tumor immunity and the development of therapeutic vaccines. We describe here evidence for the existence of SV40 Tag-specific immune responses in patients with MPM whose tumors express Tag. Humoral immunity was demonstrated by the detection of IgG titers against Tag in serum samples from 1/3 of patients examined. CTLs were generated from the peripheral blood of an HLA-A2+ MPM patient with a synthetic peptide representing an HLA-A2 binding epitope in SV40 Tag. The CTLs demonstrated epitope fine specificity, in that other peptides from SV40 Tag and a peptide from influenza virus were not recognized in the context of HLA-A2. Moreover, the CTLs were capable of recognizing mesothelioma tumor cells that expressed SV40 Tag, in an MHC class I restricted manner.

Cancer immunotherapy: avoiding the road to perdition

The hypothesis that human cancers express antigens that can be specifically targeted by cell mediated immunity has become a scientifically justifiable rationale for the design and clinical testing of novel tumor-associated antigens (TAA). Although a number of TAA have been recognized and it has been suggested that they could be useful in the immunological treatment of cancer, the complexity of human beings leads us to reflect on the need to establish new criteria for validating their real applicability. Herein, we show a system level-based approach that includes morphological and molecular techniques, which is specifically required to improve the capacity to produce desired results and to allow cancer immunotherapy to re-emerge from the mist in which it is currently shrouded.

Surrogate Tumor Antigen Vaccination Induces Tumor-Specific Immunity and the Rejection of Spontaneous Metastases

The nonimmunogenic 4T1 murine mammary carcinoma model and a model surrogate tumor antigen (sTA) were employed to explore the possibility of inducing tumor-specific immunity through active immunization in the absence of defined tumor-associated antigens. Immunization of naive mice with protein-based sTA resulted in protection from s.c. challenge, with 4T1 modified to express the sTA (4T1.sTA), or from a sTA-expressing unrelated tumor cell line (mKSA). Immunization had no effect on parental 4T1 tumor growth or the formation of parental 4T1 spontaneous lung metastases. Mice that were sTA immunized and successfully rejected 4T1.sTA challenge also rejected a subsequent challenge in the contralateral flank with parental 4T1 and strikingly prevented the formation of spontaneous parental 4T1 lung metastases. The rejection of parental 4T1 seemed to be specific for and associated with unknown 4T1 tumor-associated antigens, because rejection of mKSA did not induce cross-protection against a challenge with parental 4T1. To evaluate the effect of this vaccine approach on established disease, mice were simultaneously challenged on day 0 with 4T1.sTA and parental 4T1 in contralateral flanks and then immunized on days 3, 10, 17, and 24 with sTA protein. Tumor growth and metastasis were delayed in four of five animals, and 20% (2 of 5) of the animals were tumor free at the completion of the experiment. Together, these data suggest that prior vaccination with a sTA followed by inoculation with poorly immunogenic tumor cells modified to express the sTA activates determinant spreading and the induction of systemic tumor immunity resulting in indigenous tumor rejection.

TPD52 represents a survival factor in ERBB2-amplified breast cancer cells

TPD52 and ERBB2 co‐expression has been persistently reported in human breast cancer and animal models of this disease, but the significance of this is unknown. We identified significant positive associations between relative TPD52 and ERBB2 transcript levels in human diagnostic breast cancer samples, and maximal TPD52 expression in the hormone receptor (HR)‐ and ERBB2‐positive sub‐group. High‐level TPD52 expression was associated with significantly reduced metastasis‐free survival, within the overall cohort (log rank test, P = 8.6 × 10−4, n = 375) where this was an independent predictor of metastasis‐free survival (hazard ratio, 2.69, 95% confidence interval 1.59–4.54, P = 2.2 × 10−4, n = 359), and the HR‐ and ERBB2‐positive sub‐group (log rank test, P = 0.035, n = 47). Transient TPD52 knock‐down in the ERBB2‐amplified breast cancer cell lines SK‐BR‐3 and BT‐474 produced significant apoptosis, both singly and in combination with transient ERBB2 knock‐down. Unlike ERBB2 knock‐down, transient TPD52 knock‐down produced no reduction in pAKT levels in SK‐BR‐3 or BT‐474 cells. We then derived multiple SK‐BR‐3 cell lines in which TPD52 levels were stably reduced, and measured significant inverse correlations between pERBB2 and TPD52 levels in viable TPD52‐depleted and control cell lines, all of which showed similar proliferative capacities. Our results therefore identify TPD52 as a survival factor in ERBB2‐amplified breast cancer cells, and suggest complementary cellular functions for TPD52 and ERBB2.

A growing role for hypertrophy in senescence.

Numerous observations support the existence of senescence factors in yeast. Historically, the asymmetric propagation and accumulation of extra-chromosomal ribosomal DNA circles (ERCs) has been proposed to fulfill this function. On the other hand, several recent papers have re-invigorated the discussion of a potential role for cell size and/or hypertrophy in yeast senescence. While studies have revealed evidence both in favor of and against the hypertrophy model, the prevalent dogma largely discounts a potential role for cell size in the control of cellular lifespan. However, new results not only demonstrate a correlation between cell size and senescence, but allude to a causative role of cell size and hypertrophy in aging. In particular, the degree of hypertrophy, as determined by the rate of cell growth per generation, appears to function as a major determinant of cellular lifespan. Herein, in light of these new data, we examine the recent debate regarding a potential role for cell size in yeast aging, address criticisms of this model, and suggest that the balance is tipping in favor of hypertrophy having a causative role in aging, albeit not as the sole “aging factor.”

Overexpressed oncogenic tumor-self antigens

Overexpressed tumor-self antigens represent the largest group of candidate vaccine targets. Those exhibiting a role in oncogenesis may be some of the least studied but perhaps most promising. This review considers this subset of self antigens by highlighting vaccine efforts for some of the better known members and focusing on TPD52, a new promising vaccine target. We shed light on the importance of both preclinical and clinical vaccine studies demonstrating that tolerance and autoimmunity (presumed to preclude this class of antigens from vaccine development) can be overcome and do not present the obstacle that might have been expected. The potential of this class of antigens for broad application is considered, possibly in the context of low tumor burden or adjuvant therapy, as is the need to understand mechanisms of tolerance that are relatively understudied.