by: Harrison P. Nguyen, Peter L. Rady, Dr. Stephen K. Tyring
Abstract
Merkel Cell Carcinoma (MCC) is a highly aggressive skin malignancy hypothesized to affect Merkel Cells. Recently, the cancer was determined to have an infectious origin, a novel human DNA polyomavirus named Merkel Cell Polyomavirus (MCpyV). MCpyV expresses two non-structural proteins, the Large and Small T Antigens, which have been previously implicated in viral replication and survival. However, the Large T Antigen is found to harbor mutations prematurely truncating the MCpyV helicase and inhibiting its viral replication capability. Therefore, we propose the vital role of the Small T Antigen in the pathogenesis of the carcinoma. Here we review the hypothesized pathogenesis of the disease as well as the basic viral mechanism of the newly discovered virus.
Background
First discovered in 1875 by Friedrich Merkel, Merkel cells are present in the skin of all known vertebrates. Mice lacking the gene (Atoh1) necessary to produce Merkel cells fail to resolve fine spatial details,1 and thus, Merkel cells are hypothesized to function in the touch discrimination of shapes and textures. Recently, Merkel cells were shown to have an epidermal origin, rather than the previously hypothesized neural crest origin.2 In humans, Merkel cells are distributed along the basal zone of the epidermal, adnexal, and mucous membrane epithelium. Nevertheless, immunohistochemical analysis of Merkel cells shows staining for both epithelial and neuroendocrine markers. Merkel cells are oval-shaped, have an indented nucleus, and possess desmosomes that connect them with neighboring keratinocytes. In 1972, Cyril Toker first described an unusual, highly aggressive skin tumor with electron-dense neurosecretory granules. Since Merkel cells are the only cutaneous cells to form neurosecretory granules, the neuroendocrine carcinoma was attributed to the Merkel cells and subsequently named Merkel Cell Carcinoma (MCC). MCC is characterized by a painless, firm, red hemispherical tumor with a smooth, shiny surface that grows rapidly over a period of weeks to months.
Epidemiology
The American Cancer Society estimated 1,500 cases of MCC in the U.S. for 2008. This figure tripled the number of incidences reported twenty years ago, owing to improvements in methods of detection.3 Although the reported incidence remains ~65 times less than that of melanoma, MCC is twice as lethal; one in every three MCC patients will die from the malignancy.4 The mean age of the patients at time of diagnosis is ~70 years old.5 A strong link exists between MCC and ultraviolet light exposure. Regional incidence rates of MCC increase with sun exposure as measured by the UVB solar index.6 In particular, incidence of MCC is highest at equatorial latitudes. In one report, 81% of primary tumors occur on sun-exposed skin with 36% located on the face, the most sun-exposed anatomical site.7 Additionally, Caucasians have the greatest risk for developing the cancer. More notably, there is a clear association between MCC and immunosuppression. Chronically immunosuppressed individuals are 15 times morelikely to develop the malignancy than are age-matched controls. 4 For instance, MCC occurs more frequently in HIV and organ transplant patients (12/100,000/year) 5, and MCC is more lethal in immunosuppressed individuals with a mortality rate of up to 56%.7 Interestingly, there have been several documented cases of MCC regression following restoration of immune function. 8
Histology
Currently, the preliminary diagnosis is made on the basis of histopathology. The tumor has irregular margins, and its cells are arranged in strands or nests.9 Significant spacing between cells is typical, indicating a lack of cell-cell adhesion. As seen in many other cancers, the mitotic index in MCC tumors is very high, including many atypical mitoses.For definitive diagnoses, immunohistochemical analysis is usually required. Staining for cytokeratin 20 indicates a local aggregation of these filaments in a perinuclear dot-like pattern (Figure 1).
Pathogenesis
Currently, understanding of the molecular basis of MCC is still very limited. Because it is common in epithelial cancers, the well-described mitogen-activated kinase (MAPK) pathway has been extensively studied in MCC. As a result, it is clear how a mutation in this pathway can lead to transformation and especially immortalization. (MAPK traditionally plays a key role in many cell processes, including proliferation, suppression of apoptosis, migration, and differentiation;5 however, in several studies on MCC, little evidence has accumulated for a role of the MAPK pathway in tumorigenesis. Interestingly, traditional mutants in the MAPK pathway, such as the receptor tyrosine kinase c-kit and the ERK protein, which underlie other epithelial cancers are normal in MCC. 11,12 Although the MAPK pathway as a whole is generally inactive, a study demonstrated that inhibition of the farnesylation of the Ras protein, a particular component of the MAPK pathway, is sufficient to suppress MCC tumor growth in mice.13 The results of these studies taken together suggest the relevance of another Ras-regulated signal pathway involving the class 1 phosphoinositide 3 kinase (PI3K) and the Akt kinase. A downstream target of the PI3K/Akt pathway is the tumor suppressor p53. Induction of p53 accompanies apoptosis induced by Ras inhibition in MCC tumors.13 Although studies have indicated inconclusive evidence for the mutation of p53, the aforementioned finding that induction of p53 accompanies tumor suppression suggests that p53 expression and stability is related to MCC tumorigenesis, most likely through a downstream target of p53 in MCC progression. Protein Rb1 is a downstream target of p53 that functions as a key molecule in gene expression promoting the G1/S transition, and its demonstrated impact in virtually all cancers points to a significant role of Rb1 in MCC as well. In its hypophosphorylated state, Rb1 prevents cell cycle progression by inhibiting the E2F transcription factor. To enter S phase, cyclin-dependent kinases that are themselves indirectly regulated by p53 phosphorylate Rb and thus inactivate it, releasing E2F for cell cycle progression. Several mechanisms can allow Rb to mutate or become inactive, contributing to immortalization.5
Discovery of an infectious origin
With strong evidence supporting a correlation between immunosuppression and MCC, researchers long have hypothesized an infectious origin for the cancer. The mystery was partially unraveled in January 2008 when Feng et al. at the University of Pittsburgh identified a novel human DNA polyomavirus, which the authors named Merkel Cell Polyomavirus (MCpyV), in the full-genome sequencing of MCC samples. The authors developed a technique known as digital transcriptome subtraction (DTS), in which all mRNAs from a tumor are reverse transcribed into cDNA and then compared to the human genome. All human sequences are “subtracted,” and the remaining transcripts are assumed to be non-human sequences. Feng et al. conducted DTS with four MCC tumors, of which 99.4% of the sequences aligned closely with known human transcripts, and they established that one sequence common to all of the four tumors carried high sequence identity with the human BK polyomavirus antigen. Following extension and amplification, the authors identified the integration of a novel virus that has now been formally named MCpyV. To determine if MCpyV had a causal role, the researchers then screened ten MCC tumors for MCpyV using PCR. Seven were strongly positive and one was deemed weakly positive. Subsequent studies testing diverse populations have confirmed a ~80% presence rate of MCpyV DNA in MCC tumors, suggesting that MCpyV is a likely cause of MCC, although this has yet to be definitively confirmed.15, 16 Although all MCpyV tumors do not contain MCC, cancers that show presence of the virus are correlated with an increased potential for metastasis.17 Furthermore, based on Southern hybridization using a MCpyV DNA probe, Feng et al. determined that five of the eight MCpyV positive tumors contained monoclonal integration of the viral genome, suggesting that integration of the MCpyV genome occurs before metastases. In conjunction with the high frequency of MCpyV in MCC, this finding strongly indicates that MCpyV plays a causal role in tumorigenesis.
Polyomaviruses are small (40-50 nanometers in diameter), non-enveloped, circular, double-stranded DNA viruses. MCpyV is the sixth member of the Polyomaviridae genus that has been identified to infect humans. Polyomaviruses are highly species-specific and are thought to co-evolve with the organism that they infect. Their genomes are divided into three regions: early, late, and regulatory (Figure 2). The early region is expressed early in virus infection and continues during the late stage of infection. Importantly, the early region encodes non-structural proteins, namely the Large Tumor (T) Antigen and the Small Tumor (T) Antigen. The late region is expressed during and after genome replication and encodes structural proteins, in particular VP 1-3. The regulatory region contains transcriptional promoters and enhancers as well as the origin of replication. Because their genomes are rather basic, polyomaviruses rely on the host cell’s machinery for replication. The early-expressed genes bind to host proteins forcing the cell into S phase and thus facilitating viral replication. In previously discovered polyomaviruses, the Large T Antigen plays many diverse roles in viral replication. It is composed of three regions. Region 1 is essential in virion assembly, viral DNA replication, transcriptional control, and oncogenic transformation. Region 2 is important for host cell transformation as it contains an amino acid sequence that is important for binding the tumor suppressor pRB. Region 3 binds p53, most likely to promote cell growth and to facilitate host cell entry into S phase. In other polyomaviruses, the Large T Antigen plays an indispensable role in viral replication and survival. However, Shuda et al. identified MCC tumor-derived Large T Antigens to harbor mutations prematurely truncating the MCpyV LT helicase (22), which was confirmed by other publications (10) (Figure 3). The authors showed that with this truncation, the MCpyV Large T Antigen did not possess the capacity to replicate viral DNA but still preserved the ability to bind pRB. Thus, the MCpyV Large T Antigens is most likely still essential to the development of the carcinoma, but it does not contain the cell-lethal effects similar to the Large T Antigens of other polyomaviruses.8
Small T Antigen
With the interesting mutation and subsequent loss of function of the MCpyV Large T Antigen, questions arise as to how MCpyV can conquer host cells and replicate to infect more cells. Thus, the lab of Stephen Tyring at University of Texas at Houston Health Sciences Center proposes to focus on the other oncoprotein of interest, the Small T Antigen. In other polyomaviruses, the Small T Antigen plays a reduced, supplementary role to the Large T Antigen, but MCpyV could present a novel case where the Small T Antigen is the predominant pro-carcinogenetic component. The N-terminal sequence of the Small T Antigen is highly conserved among polyomaviruses, containing a heptapeptide region involved in cell replication. In addition, a J region (also present in the Large T Antigen) functions as a chaperone.18 However, the middle region of the Small T Antigen carries high variability, and consequently, its functionality in MCpyV is largely unknown. Most likely, it binds and inactivates protein phosphatase 2A (PP2A), an important biological enzyme for cells, and thus promotes S phase entry for the virus. In other polyomaviruses, Small T Antigen inhibition of PP2A is necessary for ST-mediated transformation to occur19(Figure 4).
PP2A refers to a family of serine-threonine phosphatases in eukaryotic cells. PP2A is composed of three subunits, each with several isoforms, allowing over 100 known PP2A complexes to exist. Particular PP2A complexes have characteristic substrates and functions within the cell, making it very difficult to establish the molecular mechanisms used by the Small T Antigen to induce transformation.20
The MCpyV Small T Antigen probably assumes a larger role in cell transformation and, in particular, replication, a process that was left unexplained given the mutation in the Large T Antigen. Thus, our lab sets out to elucidate the processes of the MCpyV Small T Antigen particularly binding partners and its behavior in a host cell environment. We already have shown that the Small T Antigen is highly conserved among MCC tumors containing the MCpyV genome, and we hope that information regarding the behavior of the protein will answer important questions regarding the pathogenesis of Merkel Cell Carcinoma.
The proposed work is a stepping stone in the struggle to understand and cure cancer. Although its prevalence is dwarfed by its melanoma and non-melanoma counterparts, Merkel Cell Carcinoma is rapidly growing in incidence rates and its 33% fatality rate presents a rather bleak outlook for the unfortunate individuals who are diagnosed with the disease. This research offers hope for understanding the mechanism by which the viral origin is able to transform and immortalize cell targets, subsequently leading to rapid metastases. The implications of this research are farreaching; knowledge into the mechanism of infection and viral survival is expected to shed light on questions relating to virology, Merkel cell function, and cancer in general.
Harrison Nguyen is a sophomore Cognitive Sciences major at Hanszen College. He is primarily interested in infectious carcinogenesis, and he works in the mucocutaneous laboratory at University of Texas Health Sciences Center under the guidance of Dr. Stephen Tyring. His laboratory is specifically studying the role of the Small Tumor Antigen in Merkel Cell Carcinogenesis. Harrison aspires to pursue his passion of medicine in the field of dermatological oncology as a research-physician.
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