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Human X-box binding protein-1 (XBP1) is an alternatively spliced transcription factor that participates in the unfolded protein response (UPR), a stress-signaling pathway that allows cells to survive the accumulation of unfolded proteins in the endoplasmic reticulum lumen. We have previously demonstrated that XBP1 expression is increased in antiestrogen-resistant breast cancer cell lines and is coexpressed with estrogen receptor alpha (ER) in breast tumors. The purpose of this study is to investigate the role of XBP1 and the UPR in estrogen and antiestrogen responsiveness in breast cancer. Overexpression of spliced XBP1 [XBP1(S)] in ER-positive breast cancer cells leads to estrogen-independent growth and reduced sensitivity to growth inhibition induced by the antiestrogens Tamoxifen and Faslodex in a manner independent of functional p53. Data from gene expression microarray analyses imply that XBP1(S) acts through regulation of the expression of ER, the antiapoptotic gene BCL2, and several other genes associated with control of the cell cycle and apoptosis. Testing this hypothesis, we show that overexpression of XBP1(S) prevents cell cycle arrest and antiestrogen-induced cell death through the mitochondrial apoptotic pathway. XBP1 and/or the UPR may be a useful molecular target for the development of novel predictive and therapeutic strategies in breast cancer.

To gain insight into the role of genomic alterations in breast cancer progression, we conducted a comprehensive genetic characterization of a series of four cell lines derived from MCF10A. MCF10A is an immortalized mammary epithelial cell line (MEC); MCF10AT is a premalignant cell line generated from MCF10A by transformation with an activated HRAS gene; MCF10CA1h and MCF10CA1a, both derived from MCF10AT xenografts, form well-differentiated and poorly-differentiated malignant tumors in the xenograft models, respectively. We analyzed DNA copy number variation using the Affymetrix 500 K SNP arrays with the goal of identifying gene-specific amplification and deletion events. In addition to a previously noted deletion in the CDKN2A locus, our studies identified MYC amplification in all four cell lines. Additionally, we found intragenic deletions in several genes, including LRP1B in MCF10CA1h and MCF10CA1a, FHIT and CDH13 in MCF10CA1h, and RUNX1 in MCF10CA1a. We confirmed the deletion of RUNX1 in MCF10CA1a by DNA and RNA analyses, as well as the absence of the RUNX1 protein in that cell line. Furthermore, we found that RUNX1 expression was reduced in high-grade primary breast tumors compared to low/mid-grade tumors. Mutational analysis identified an activating PIK3CA mutation, H1047R, in MCF10CA1h and MCF10CA1a, which correlates with an increase of AKT1 phosphorylation at Ser473 and Thr308. Furthermore, we showed increased expression levels for genes located in the genomic regions with copy number gain. Thus, our genetic analyses have uncovered sequential molecular events that delineate breast tumor progression. These events include CDKN2A deletion and MYC amplification in immortalization, HRAS activation in transformation, PIK3CA activation in the formation of malignant tumors, and RUNX1 deletion associated with poorly-differentiated malignant tumors.

Merkel cell carcinoma (MCC) is a rare but devastating skin disease that is increasing in incidence within the United States. The poor prognosis of MCC patients and limited understanding of MCC pathogenesis warrants innovative treatments to control MCC. Several lines of evidence have pointed to Merkel cell polyomavirus (MCPyV) as the etiological agent of MCC. In particular, the amino terminus of MCPyV large T antigen (LT) (aa1-258) is expressed in all MCPyV-positive tumors and plays an important role in MCC oncogenesis, rendering it an ideal therapeutic target for vaccination. In the current study, we developed a DNA vaccine encoding MCPyV LT aa1-258 (pcDNA3-LT). Within our pcDNA3-LT DNA vaccine, we identified that MCPyV LT aa136-160 likely contains an LT-specific CD4+ T helper epitope. We have also created an LT-expressing B16/LT tumor model using B16, a murine melanoma cell line, to characterize the potency of our DNA vaccine. Using this tumorigenic B16/LT tumor model, we found that pcDNA3-LT DNA vaccine generates antitumor effects mainly mediated by CD4+ T cells against B16/LT tumors in vaccinated C57BL/6 mice. Thus, immunotherapy using pcDNA3-LT DNA vaccine may represent a promising approach for the control of MCPyV-associated lesions. The B16/LT tumor model further serves as a useful model for testing various vaccine strategies against MCC.

Abstract. Interferon regulatory factor-1 (IRF-1), human X-box binding protein-1 (hXBP-1), nuclear factor kappa B p65 (NFκB p65) and nucleophosmin (NPM) have been implicated in a signaling network of endocrine responsiveness. Expression of these proteins was measured by immunohistochemistry in tissue microarrays of 54 breast tumors. Correlations between each protein and established prognostic markers were assessed by Spearman's rank order correlation coefficient and partial correlation coefficient analyses. Moderate/strong staining is seen for hXBP-1 (79% of tumors) and NFκB p65 (57%). NPM exhibits nuclear staining (95%); IRF-1 exhibits both cytosolic (IRF-1c; 90%) and nuclear staining (IRF-1n; 51%). IRF-1c is associated with age (p=0.034); IRF-1n and PgR expression are correlated (p=0.014). NFκB p65 shows a borderline association with S phase (p=0.062). Coexpression of IRF-1c and hXBP1 (p=0.001), , and hXBP-1 and NFκB (p=0.018) is observed. An inverse correlation exists between IRF-1n and NFκB (p=0.034). All four proteins are detected in breast tumors and their expression patterns support their role(s) in a key signaling network.

BackgroundMerkel cell polyomavirus (MCPyV) is a DNA virus expressing transcripts similar to the large T (LT) and small T (ST) transcripts of SV40, which has been implicated in the pathogenesis of Merkel cell carcinoma (MCC), a rare and highly aggressive neuroendocrine skin cancer. MCPyV LT antigen expression was found to be a requirement for MCC tumor maintenance and ST protein also likely contributes to the carcinogenesis of MCC. Previously, we have identified the probable immunodominant epitope of MCPyV LT and developed a DNA vaccine encoding this epitope linked to calreticulin. The LT-targeting DNA vaccine generated prolonged survival, decreased tumor size and increased LT-specific CD8+ T cells in tumor-bearing mice.ResultsIn this study, we developed a MCPyV ST-expressing tumor cell line from B16 mouse melanoma cells. We then utilized this ST-expressing tumor cell line to test the efficacy of a DNA vaccine encoding ST. In ST-expressing tumor-bearing mice, this vaccine, pcDNA3-MCC/ST, generated a significant number of ST antigenic peptide-specific CD8+ T cells and experienced markedly enhanced survival compared to mice vaccinated with empty vector.ConclusionsThe formation of an effective vaccine against MCPyV has the potential to advance the field of MCC therapy and may contribute to the control of this severe malignancy through immunotherapy. Both of the innovative technologies presented here provide opportunities to develop and test MCPyV-targeted therapies for the control of Merkel cell carcinoma.

BackgroundMerkel cell carcinoma (MCC) is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV). The MCPyV-encoded large T (LT) antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT) encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT), as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells.ResultsThe present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the immunodominant LT epitope as aa19-27 (IAPNCYGNI) and found that it is H-2kb-restricted.ConclusionThe results of this study can facilitate the development of other modes of MCC treatment such as peptide-based vaccines and adoptive transfer of LT-specific CD8+ T cells. Likewise, the MCC DNA vaccine has great potential for clinical translation as the immunologic specificity is high and the treatment strategy can be exported to address other virus-induced tumors.

This exploratory study investigates former international students’ experiences pursuing permanent status with the use of primary data from interviews with five individuals. Guided by the question, “what characterizes former international students’ trajectories to permanent residence” and based on the understanding that discourses of exclusion and control inform immigration policies today (Fobear, 2014), personal experiences are explored as realities of temporariness in which subjects are contained by the following forms of regulation: time limits, employment specificity, and temporary legal status. Anthony Giddens’ structuration theory is employed to showcase participants as “knowledgeable” (Sewell, 1992:4) and reflexive agents (Turner, 1986); how they persevere and negotiate their way to permanent residence by enacting creative strategies and enduring the emotional labour that characterize their search for and securing of ‘skilled’ employment while mitigating the immediate need for income, in reframing their mindsets and in their reflections upon the meaning of their pursuits for permanence.