Jianning Mao

Autoantibody Signatures Involving Glycolysis and Splicesome Proteins Precede a Diagnosis of Breast Cancer among Postmenopausal Women

We assessed the autoantibody repertoire of a mouse model engineered to develop breast cancer and the repertoire of autoantibodies in human plasmas collected at a preclinical time point and at the time of clinical diagnosis of breast cancer. In seeking to identify common pathways, networks, and protein families associated with the humoral response, we elucidated the dynamic nature of tumor antigens and autoantibody interactions. Lysate proteins from an immortalized cell line from a MMTV-neu mouse model and from MCF7 human breast cancers were spotted onto nitrocellulose microarrays and hybridized with mouse and human plasma samples, respectively. Immunoglobulin-based plasma immunoreactivity against glycolysis and spliceosome proteins was a predominant feature observed both in tumor-bearing mice and in prediagnostic human samples. Interestingly, autoantibody reactivity was more pronounced further away than closer to diagnosis. We provide evidence for dynamic changes in autoantibody reactivity with tumor development and progression that may depend, in part, on the extent of antigen-antibody interactions.

An Autoimmune Response Signature Associated with the Development of Triple-Negative Breast Cancer Reflects Disease Pathogenesis

The repertoire of antigens associated with the development of an autoimmune response in breast cancer has relevance to detection and treatment strategies. We have investigated the occurrence of autoantibodies associated with the development of triple-negative breast cancer (TNBC) in the before diagnosis setting and in samples collected at the time of diagnosis of TNBC. Lysate arrays containing protein fractions from the TNBC MDA-MB-231 cell line were hybridized with TNBC plasmas from the Womens Health Initiative cohort, collected before clinical diagnosis and with plasmas from matched controls. An immune response directed against spliceosome and glycolysis proteins was observed with case plasmas as previously reported in estrogen receptor(+) breast cancer. Importantly, autoantibodies directed against networks involving BRCA1, TP53, and cytokeratin proteins associated with a mesenchymal/basal phenotype were distinct to TNBC before diagnosis samples. Concordant autoantibody findings were observed with mouse plasma samples collected before occurrence of palpable tumors from a C3(1)-T triple negative mouse model. Plasma samples collected at the time of diagnosis of stage II TNBC and from matched healthy controls were subjected to proteomic analysis by mass spectrometry to identify Ig-bound proteins yielding a predominance of cytokeratins, including several associated with a mesenchymal/basal phenotype among cases compared with controls. Our data provide evidence indicative of a dynamic repertoire of antigens associated with a humoral immune response reflecting disease pathogenesis in TNBC.

Abstract 1352: Identifying pre-diagnostic breast cancer antigens in transgenic mouse mammary tumor models for preventative vaccine development

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Preventative vaccine therapy may benefit women at high risk for breast cancer but currently it is impossible to identify women who will develop breast cancer prior to tumor development to identify initiating cancer antigens. Immunocompetent transgenic mouse mammary tumor models that are genetically similar to human breast cancer subtypes may identify pre-invasive antigens because mice can be followed longitudinally. Comparing tumor growth characteristics in two mouse models (TgMMTV-neu is genetically similar to luminal B breast cancer and C3(1)tag is genetically similar to triple negative breast cancer) demonstrated that, similar to triple negative breast cancer, C3(1)Tag mice develop tumors earlier and tumors grow more rapidly than TgMMTV-neu tumors. Furthermore, C3(1)Tag tumors have increased CD8+/CD4+ ratio (p<0.05) similar to human triple negative tumors. Using these two mouse models, we identified putative pre-invasive breast cancer antigens present prior to tumor development by serologic analysis of recombinant cDNA expression libraries and serological analysis of chip-arrayed proteins. We identified 65 pre-invasive antigens that were present in mice that would develop cancer but not parental control mice. The goal of this study was to identify which of the 65 antigens were required for human breast cancer survival with the goal to develop a preventative multi-antigen polyepitope breast cancer vaccine. We chose the pre-invasive antigens necessary for human breast cancer tumor cell survival using a high throughput siRNA screen evaluating for increased apoptosis and decreased cell survival in either HER2 positive or triple negative human breast cancer cell lines with decreased expression of the target protein. Five of the antigens were essential for human breast cancer cell survival: VPS35, SERBP1, ARPC2, PDIA6, and KRT8. All of these genes have roles in human cancer progression, and KRT8, SERBP1, and PDIA6 have identified roles in breast cancer pathogenesis. After designing human MHC class II peptides for each of these targets that cover at least 25% of the protein, we evaluated implanted tumor inhibition in the mouse models. Vaccination VPS35 peptides inhibited tumor growth by 47% (p<0.0001), vaccination with ARPC2 peptides inhibited tumor growth by 54% (p<0.0001), and vaccination with SERBP1 peptides inhibited tumor growth by 61% (p<0.0001) in the TgMMTV-neu mice. In C3(1)Tag mice, vaccination with VPS35 peptides inhibited tumor growth by 39% (p<0.0001) and vaccination with SERBP1 peptides inhibited tumor growth by 59% (p<0.0001) but vaccination with ARPC2 peptides did not inhibit tumor growth. These studies have demonstrated that mouse models can be used to identify pre-invasive breast cancer antigens. Further studies will evaluate the use of vaccines containing epitopes from several of the antigens to prevent spontaneous tumors in these mouse models. Citation Format: Sasha E. Stanton, Ekram Gadd, Lauren Rastetter, James Annis, Jianning Mao, John Ladd, Samir Hanash, Mary L. Disis. Identifying pre-diagnostic breast cancer antigens in transgenic mouse mammary tumor models for preventative vaccine development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1352. doi:10.1158/1538-7445.AM2015-1352

Vaccination with pre-diagnostic tumor antigens but not established tumor antigens inhibits spontaneous tumorigenesis in TgMMTV-neu mice

Breast cancer is immunogenic and proteins expressed in breast cancer stimulate both an antibody and a T-cell immune response. Cancer related proteins that are aberrant enough to trigger early immune recognition might be ideal targets for a vaccine aimed at preventing the development of breast cancer. Antigen discovery has focused on the identification of immunogenic proteins in tumor bearing individuals; in this study we have used SEREX and a transgenic mouse model to identify immunogenic proteins in animals prior to the development of cancer. These antigens may represent the earliest alterations of the malignant transformation. We identified 6 antigens that were present in mice prior to the development of mammary cancer (Pdhx, Otud6b, Stk39, Zpf238, Lgals8, and Vps35). These proteins were associated with inflammation, autoimmunity, and cellular homeostasis, were highly homologous to human, and were overexpressed in ductal carcinoma in situ and invasive breast cancer in women. We questioned whether these antigens could mediate anti-tumor immunity. When TgMMTV-neu mice (n=5/group) were vaccinated with the individual antigens, vaccination with Pdhx inhibited tumor growth by 62.1%, Otud6B inhibited tumor growth by 23.5%, and Stk39 inhibited tumor growth by 50.3% as compared to empty vector vaccinated control at 27 weeks (p<0.001 for each individual antigen as compared to empty vector). Spontaneous tumorigenesis was inhibited in TgMMTV-neu mice (n=20 mice/group) vaccinated with a panel of three of the early tumor antigens (Pdhx, Otud6b, and Stk39). This panel of antigens inhibited tumor growth by 34.8% by 37 weeks as compared to vector vaccinated mice (p=0.02). Tumorigenesis was similar to empty vector when animals were vaccinated with a panel of antigens that were discovered by using sera derived from animals with established tumors previously identified in our laboratory (Arhgef2, Swap70, and GSN). At 37 weeks the mice vaccinated with the established tumor vaccine had similar growth as compared to vector-vaccinated mice (p=0.69) but 31.1% more growth than the early tumor antigen vaccinated mice (p=0.005). These data suggest vaccination against a panel of pre-invasive breast tumor proteins inhibit tumorigenesis in mice unlike vaccination with a panel of established tumor proteins, suggesting that these pre-invasive antigens are better targets for a preventative vaccine. Future studies will address if these antigens elicit T-cell responses in patients with high-risk breast lesions.

Abstract 2491: Dynamics changes in antigen-autoantibody profiles involving glycolysis and spliceosome proteins precede a diagnosis of ER+ and triple negative breast cancer among post-menopausal women.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC We assessed the circulating autoantibody repertoire in breast cancer using genetically engineered mouse models and human plasmas collected at a pre-clinical time point and at the time of clinical diagnosis of breast cancer. We aimed to identify common pathways, networks and protein families associated with the humoral response to tumors and to elucidate the dynamic nature of tumor antigens and autoantibody interactions in ER+ and triple negative breast cancer. Lysate proteins from immortalized mouse cell lines from MMTV-neu and C3(1)-T mouse models and from the MCF7 and MDA-MB-231 human breast cancer cell lines were spotted onto nitrocellulose microarrays and hybridized with mouse and human plasma samples, respectively. Ig-based plasma immunoreactivity against glycolysis and spliceosome proteins was a predominant feature observed both in MMTV-neu tumor bearing mice and in pre-diagnostic ER+ human samples. A cytoskeletal signature was uniquely observed in C3(1)-T mice and in human patients with triple negative breast cancer. We provide evidence for dynamic changes in autoantibody reactivity and in antigen-antibody interactions with tumor development and progression. Citation Format: Jon Ladd, Melissa Johnson, Timothy Chao, Alice Chin, Sharon Pitteri, Jianning Mao, Lynn M. Amon, Martin McIntosh, Paul Lampe, Christopher Li, Ross Prentice, Nora Disis, Samir Hanash. Dynamics changes in antigen-autoantibody profiles involving glycolysis and spliceosome proteins precede a diagnosis of ER+ and triple negative breast cancer among post-menopausal women. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2491. doi:10.1158/1538-7445.AM2013-2491

Abstract P5-01-09: Early tumor antigens discovered in TgMMTV-neu mice may provide targets for early breast cancer diagnosis and prevention

Breast cancer is immunogenic and breast tumor proteins stimulate both an antibody and a T cell immune response. Identification of cancer related proteins that become immunogenic prior to the clinical development of cancer may allow the use of humoral immunity to identify those “exposed” to the cancer phenotype. Alternatively, these proteins that become aberrant enough to trigger such early immune recognition may be ideal targets for a vaccine aimed at preventing the development of breast cancer. We used the TgMMTV-neu mouse model to discover immunogenic proteins i.e. proteins expressed in pre-invasive breast cancer. The mammary tumors in these mice are genotypically similar to human luminal breast cancer and can be evaluated longitudinally, allowing for collection of pre-diagnostic sera prior to tumor development to use for antigen discovery. We identified 6 antigens that were present in mice prior to the development of mammary cancer (Pdhx, Otud6b, Stk39, Zpf238, Lgals8, and Vps35). These proteins were associated with inflammation, autoimmunity, and cellular homeostasis. In mouse validation cohorts, detecting IgM and IgG antibody responses against a panel of three “pre-diagnostic” tumor antigens discriminated pre-diagnostic sera from non-transgenic control sera with an AUC of 0.924. We next evaluated samples obtained from the Womens Health Initiative Study and demonstrated women with autoantibodies to the human homologues of these proteins. IgM and IgG autoantibodies to the “pre-diagnostic” antigen panel could discriminate the samples of women who eventually developed breast cancer from matched controls. The discriminatory potential of the pre-diagnostic autoantibodies was enhanced if samples were collected more than 5 months prior to diagnosis (AUC 0.68; CI 0.565-0.787). We questioned whether these antigens, which could predict women who would eventually develop breast cancer, could mediate anti-tumor immunity. When TgMMTV-neu mice (n = 5/group) were vaccinated with the individual antigens, vaccination with Pdhx inhibited tumor growth by 62.1%, Otud6B inhibited tumor growth by 23.5%, and Stk39 inhibited tumor growth by 50.3% as compared to empty vector vaccinated control at 27 weeks (p<0.001 for each of the individual antigens as compared to empty vector). Spontaneous tumorigenesis was inhibited in TgMMTV-neu mice (n = 20 mice/group) vaccinated with a panel of three of the “pre-diagnostic” antigens (Pdhx, Otud6b, and Stk39) inhibited tumor growth by 27.3% by 37 weeks as compared to vector vaccinated mice (p<0.05). These data suggest that the same pre-invasive breast tumor proteins are found in mice and women and vaccines against these pre-invasive breast cancer proteins inhibit tumorigenesis in mice, future studies will address if these antigens elicit T-cell responses in patients with high risk breast lesions. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-09.

Abstract 750: Identification of tumor antigens in transgenic mouse model as vaccine targets for the prevention of human breast cancer

The development of a vaccine to prevent breast cancer would be facilitated if tumor antigens were identified which were associated with the initiation of the malignant transformation. Transgenic mouse models have been proven to demonstrate a similar antigenic repertoire as that seen in breast cancer patients, therefore, we propose to use these models to identify tumor antigens as vaccine targets for breast cancer prevention. We have chosen two mouse models: TgMMTV-neu and TgC3(1)-Tag for study. TgMMTV-neu mouse has a genotype similar to luminal breast cancer and the TgC3(1)-Tag model reflects a basal phenotype of breast cancer. We identified potential vaccine candidate antigens in one of two ways; (1) determining which proteins expressed by the cancer are immunogenic very early in the malignant process by screening pre-diagnostic sera for immunogenicity against tumor cDNA libraries, and (2) injecting parental animals with a syngeneic tumor cell line resulting in “tumor rejection” which we have shown previously is mediated by T cells. To date, we have identified nine pre-diagnostic tumor antigens in TgMMTV-neu mouse; Rpl5, TNFaip3/A20, Pdhx, Otud6b, Stk39, Zfp238, Dnajc10, Lgals8 and Vps35. Moreover, we have identified four rejection antigens in TgC3(1)-Tag mouse; Ddx21, Sfrs11, Edh1 and Tdg. We have initiated vaccination experiments to examine the anti-tumor effect of vaccination with plasmids encoding these antigens. We found that vaccination targeting Ddx21, Dnajc10, and Pdhx mediated tumor growth inhibition in implant models compared with a control group (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 750. doi:10.1158/1538-7445.AM2011-750

Abstract 2683: Identification of tumor antigens in neu transgenic mice as diagnostic targets for the early detection of human breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Tumor antigens, expressed early in disease, which can induce specific antibody response could serve as biomarkers for early detection of human breast cancer. However, the ability to obtain sera from women prior to the development of breast cancer is limited, which makes it difficult to identify early antigens directly from breast cancer patients. Transgenic mouse models have been proved to demonstrate similar antigen repertoire to breast cancer patients. Therefore, we propose to identify early tumor antigens in neu transgenic mice and use them as diagnostic targets for early detection of human breast cancer. We have collected serial sera from neu transgenic mice, sera collected before the animals develop palpable tumor “pre-diagnostic sera”. With serological analysis of recombinant cDNA expression libraries (SEREX) approach, we were able to identify nine tumor antigens from pre-diagnostic sera, Rpl5, TNFaip3/A20, Pdhx, Otud6b, Stk39, Zfp238, Dnajc10, Lgals8 and Vps35. These antigens demonstrated detectable IgG antibody response prior to tumor development in neu transgenic mice. IgM is the first antibody to be produced during a humoral immune response, so we questioned whether including antigen specific IgM with IgG responses could achieve a better diagnostic performance. Firstly, we analyzed receiver-operating-characteristic (ROC) curves of these antigens by IgG and IgM ELISA analysis in neu transgenic mice and 26 FVB control mice. By combining IgG and IgM ELISA, we were able to achieve a higher AUC: Otud6b and Stk39 show an AUC of 0.882, and adding Lgals8 IgG ELISA to this panel can achieve an AUC of 0.924 in discriminating cases from control mice. We documented that these autoantibodies are also detectable in the serum of women with breast cancer and then screened a panel of sera derived from the Womens Health Initiative cohort (samples obtained 6-18 months prior to the development of breast cancer) and case matched controls. We evaluated serum for the presence of IgG and IgM specific autoantibodies to Pdhx, Otud6b, and Stk39. With both IgG and IgM ELISA analysis, a panel combining the three antigens demonstrates an AUC of 0.633 in patients diagnosed within 5 month and an AUC of 0.688 in patients diagnosed within 5-18 month of the blood draw. In summary, we conclude that tumor antigens identified in neu transgenic mouse model can be used for early detection and diagnosis of breast cancer in women. The combination of antigen specific IgG and IgM antibodies may enhance the sensitivity of the assay for early detection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2011-2683

Abstract 2742: Identification of tumor antigens in neu transgenic mice may provide biomarkers useful for the early detection and diagnosis of human breast cancer

Early detection of breast cancer is crucial for better prognosis and successful treatment of the patients. Development of novel methods for screening for breast cancer is needed for early detection and diagnosis. The human immune system responds to tumor-specific antigens in the pre-malignant stage of breast cancer and produces specific antibodies, which can be detected as potential breast cancer biomarkers. Due to the limited availability of well-annotated pre-cancer and post-cancer sera from human subjects, we performed serial serum collection in a neu transgenic mouse model from the age of 10 weeks to the terminal stage of disease. Using a technique called serological screening of cDNA expression library (SEREX), a high-throughput method to rapidly screen recombinant proteins expressed in a bacteriophage-based cDNA library, we have previously identified tumor antigens in the late stage of neu breast cancers. The mouse tumors have similar immunogenicity as their human counterparts due to the fact that some of these antigens are also immunogenic in human. Indeed, we have found that one mouse antigen called gelsolin, shares 93% homology with human gelsolin and has the highest response to human serum antibody among other antigens. In an ELISA analysis using the serum samples from 50 breast cancer cases and 50 normal donors, the area-under-curve (AUC) value of the receiver-operating-characteristic (ROC) curve of gelsolin ELISA detects more than 70% of cancer patients. Using the SEREX technique, we sought to further identify the early-stage serum antibody biomarkers by comparing the pre-cancer, early stage, and late stage sera from the same mouse. We have identified three early-stage biomarkers, A20/TNFaip3, Rpl5, and Pdhx, in neu mice. In summary, we conclude that identification of tumor antigens in the neu transgenic animal model is useful for the discovery of serum antibody biomarkers for early detection and diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2742.