Merkel cell polyomavirus–negative Merkel cell carcinoma is associated with JAK‐STAT and MEK‐ERK pathway activation

Abstract Merkel cell polyomavirus (MCPyV) is monoclonally integrated into the genomes of approximately 80% of Merkel cell carcinomas (MCCs). While the presence of MCPyV affects the clinicopathological features of MCC, the molecular mechanisms of MCC pathogenesis after MCPyV infection are unclear. This study investigates the association between MCPyV infection and activation of the MEK‐ERK and JAK‐STAT signaling pathways in MCC to identify new molecular targets for MCC treatment. The clinicopathological characteristics of 30 MCPyV‐positive and 20 MCPyV‐negative MCC cases were analyzed. The phosphorylation status of MEK, ERK, JAK, and STAT was determined by immunohistochemical analysis. The activation status of the MEK‐ERK and JAK‐STAT pathways and the effects of a JAK inhibitor (ruxolitinib) was analyzed in MCC cell lines. Immunohistochemically, the expression of pJAK2 (P = .038) and pERK1/2 (P = .019) was significantly higher in MCPyV‐negative than in MCPyV‐positive MCCs. Male gender (hazard ratio [HR] 2.882, P = .039), older age (HR 1.137, P < .001), negative MCPyV status (HR 0.324, P = .013), and advanced cancer stage (HR 2.672, P = .041) were identified as unfavorable prognostic factors; however, the phosphorylation states of JAK2, STAT3, MEK1/2, and ERK1/2 were unrelated to the prognosis. The inhibition of cell proliferation by ruxolitinib was greater in MCPyV‐negative MCC cell lines than in an MCPyV‐positive MCC cell line. The expression of pERK1/2 and pMEK was higher in MCPyV‐negative than in MCPyV‐positive cell lines. These results suggest that activation of the JAK2 and MEK‐ERK pathways was more prevalent in MCPyV‐negative than in MCPyV‐positive MCC and the JAK inhibitor ruxolitinib inhibited MEK‐ERK pathway activation. Consequently, the JAK‐STAT and MEK‐ERK signaling pathways may be potential targets for MCPyV‐negative MCC treatment.


| INTRODUC TI ON
Merkel cell carcinoma (MCC) is a clinically aggressive neuroendocrine skin cancer that is associated with Merkel cell polyomavirus (MCPyV) in 80% of the cases. 1,2 MCPyV-positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. 3 The features of MCC differ depending on MCPyV status in terms of morphology, 1 prognosis, [4][5][6] and molecular features, including UV-related genomic mutations 7 and activation of the phosphatidylinositol-3kinase (PI3K)-Akt-mTOR 8,9 and Notch signaling pathways. 10 The Akt-mTOR signaling pathway provides cross talk between several signaling pathways, including JAK-STAT and MEK-ERK. JAK2 activation induces activation of several signaling pathways, including the transcription factor STAT3 and the mitogen-activated protein kinase (MAPK) pathway involving MEK and ERK kinase. 11 The JAK-STAT and MEK-ERK signaling pathways play a multitude of important biological functions in both normal and malignant cells.
Aberrant activation of these pathways has been observed in numerous cancers, including breast, lung, and head and neck carcinoma. 12 A previous study reported that pSTAT expression is associated with unfavorable outcomes in MCC. 13 The JAK-STAT signaling pathway can also directly mediate hepatitis B-driven Hepatocellular carcinoma tumorigenesis. 14 A recent study has shown that the trichodysplasia spinulosa polyomavirus (TSPyV) middle T antigen is involved in hyperactivation of the MEK-ERK-MNK1 signaling axis. 15

| Patient specimens
This study was approved by the institutional review boards at Kyushu University (#2019-030) and Tottori University (#1216).
The patient specimens were prepared as formalin-fixed, paraffinembedded (FFPE) samples. We performed immunohistochemical staining of CK20 and neuroendocrine markers such as synaptophysin, chromogranin A, and CD56, and the histological diagnoses were reconfirmed by four pathologists (TI, DN, SK, and KH). We also confirmed the negative status for thyroid transcription factor-1 (TTF-1) to distinguish MCC from lung cancer metastases. MCPyV infection status was analyzed in the previous study using quantitative PCR and immunostaining using an antibody against MCPyV large T antigen (MCPyV-LT). 8

| DNA extraction, next-generation sequencing, and bioinformatics
For next-generation sequencing, we selected cases in which the number of samples required for analysis was sufficient. The total DNA was isolated from MCC cases using the GeneRead DNA FFPE kit (Qiagen). DNA quality was tested using a Bioanalyzer 2100 with

| Statistical analysis for clinicopathological data
Clinicopathological parameters and immunohistochemical findings were analyzed based on the MCPyV status using the Wilcoxon or Fisher exact test. Correlations among expression levels, as obtained in immunohistochemical analysis, were calculated using Spearman's rank correlation coefficient. Prognostic analysis was performed using the log-rank test with the Kaplan-Meier analysis. The goodness of fit of each Cox model was evaluated using the likelihood ratio test. Data were analyzed using the SPSS software (version 20.0 J, SPSS Japan). A P-value of <.05 was considered significant.

| Western blot analysis
Cells were washed twice with PBS, centrifuged, resuspended in 2× sodium dodecyl sulfate (SDS) sample buffer, and then denatured at 97°C for 3 minutes as performed previously. 19 The supernatant of the lysate was separated via SDS-PAGE (4%-20% Mini-PROTEAN TGX gel, Bio-Rad) and electrotransferred to a polyvinylidene fluoride membrane (2.5 A, 25 V, 7 minutes) using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories). The membrane was blocked for 15 minutes in Blocking One-P (Nacalai Tesque) and incubated with primary antibodies in Hikari Solution A (Nacalai Tesque) followed by incubation with secondary antibodies and detection using Chemi-Lumi One Ultra (Nacalai Tesque). The primary antibodies used for Western blotting are summarized in Table S1.

| Prognostic factors
Follow-up information was available for patients; the average follow-up period after surgery was 22 months (range, 1-72 months).
No cases were treated with immune checkpoint inhibitors. Results of survival analysis using the Kaplan-Meier method and log-rank test are summarized in Figure 3. Higher pSTAT3 expression was associated with favorable outcomes, but this relationship was not significant (overall survival [OS], P = .417). Log-rank test results show that the expressions of pJAK2, pERK, and pMEK were not associated with prognosis. Univariate Cox proportional hazard analyses showed that male sex, old age, negative MCPyV status, and advanced stage were unfavorable prognostic factors (Table 2).

| Western blot analysis
The expression of JAK-STAT and MEK-ERK pathway proteins was as-  Figure S1). Elevated expression of pERK1/2 was inhibited by ruxolitinib in MCC13 and MCC14/2, but not in MKL-1 cells ( Figure 4C). pSTAT3 expression was higher in MCC14/2 than in MCC13 or MKL-1 cells, and this pSTAT3 overexpression was not altered by ruxolitinib treatment in MCC13 cells ( Figure 4C). JAK inhibition resulted in dose-dependent STAT3 inactivation in MKL-1 cells, but not in MCC13 cells ( Figure 4C). Notably, treatment with JAK inhibitor at 5 µM concentration for 1 day markedly increased the pSTAT3 level ( Figure S1). pSTAT1 expression was increased in MCC 14/2 cells treated with 5µM ruxolitinib at day 1 ( Figure S1). STAT1 expression was decreased by 50 µM ruxolitinib treatment in the MCC13 and MCC14/2 cell lines ( Figure 4C). Prognostic factor analysis reconfirmed the MCPyV-negative status was associated with unfavorable outcomes, as indicated in our and other previous studies. 4,21 Advanced AJCC stage is also an unfavorable prognostic factor, as previously reported. 31 Activated STAT3 positively correlates with a better prognosis in patients with colorectal carcinoma, leiomyosarcoma, and nasopharyngeal carcinoma. [32][33][34] In our study, Kaplan-Meier analysis showed that patients with higher STAT3 expression had more favorable outcomes than did those with lower expression; however, this difference was not statistically significant. The other immunohistochemical markers related to the JAK-STAT or MEK-ERK pathway were not related to prognosis.     work was presented at the 110th USCAP annual meeting.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflict of interests.