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The FOXP3 gene maps to the short arm of chromosome-X (Xp11.23) and encodes a member of the forkhead box (FOX) family of transcription factors. Characterized by a highly conserved forkhead (FKH) DNA-binding domain, FOXP3 is required for the differentiation of CD4+ CD25+ regulatory T cells (Treg), which are critical for the maintenance of immune homeostasis and self-tolerance (Gavin et al., 2007). Mutations resulting in deletion of the FKH DNA-binding domain of FOXP3 give rise to lethal autoimmune diseases in affected males (Brunkow et al., 2001). A series of recent studies have demonstrated FOXP3 expression in normal epithelial cells of the breast, prostate, and ovary (Wang et al., 2009; Zhang and Sun, 2010; Zuo et al., 2007), as well as in a wide range of cancer cells including metastatic melanoma, leukemia, pancreatic adenocarcinoma and hepatocellular carcinoma (Ebert et al., 2008; Hinz et al., 2007; Kim et al., 2011a; Quaglino et al., 2011; Wang et al., 2010), indicating that FOXP3 expression is not restricted to the Treg lineage.

The evidence generated to date suggests that FOXP3 can act as either a tumor suppressor or promoter, depending on the tumor type. Evidence that FOXP3 may function as a tumor suppressor includes the following: First, somatic inactivating mutations and deletions in FOXP3 have been observed in breast and prostate cancers (Wang et al., 2009; Zuo et al., 2007). Notably, however, a recent study in a cohort of Korean patients with prostate cancer failed to confirm this finding (Kim et al., 2011b). Second, FOXP3 has been shown to directly repress the expression of two oncogenes involved in mammary carcinogenesis, HER-2 and SKP2 (Zuo et al., 2007), as well as the c-MYC oncogene in prostate cancer (Wang et al., 2009). Finally, overexpression of FOXP3 in ovarian cancer cells inhibited proliferation, migration, and invasion, suggesting that FOXP3 may function as a tumor suppressor in this tumor type (Zhang and Sun, 2010). In contrast, FOXP3-expressing pancreatic carcinoma cells have been shown to suppress effector T cell proliferation, mimicking the effect of Treg and suggesting a role for FOXP3 in tumor-mediated immune evasion (Hinz et al., 2007).

We have recently demonstrated that FOXP3 is expressed in human melanoma biopsies and cell lines. However, whether FOXP3 functions as a tumor suppressor or promoter in melanoma is unknown. To address this, we determined the mutational status of FOXP3 in melanoma, as recently performed for human breast and prostate cancer. In this study, we investigated the mutational status of FOXP3 in 54 early passage melanoma cell lines generated in our laboratory from patients with advanced (stages III and IV) metastatic melanomas (primers and cell line information are available in Supporting information Tables S1 & S2).

Mutations in exons 3, 4, 5, 6, 10, 11, and 12 (clustering around the forkhead, repressor, and zinc finger domains) of the FOXP3 gene have previously been described in breast and prostate cancer (Wang et al., 2009; Zuo et al., 2007). To determine whether mutations in these exons also occur in melanoma, we first sequenced these exons in 54 early passage (less than 10 passages) melanoma cell lines derived from patients with advanced (stages III and IV) metastatic melanomas. We also determined the mutational status of FOXP3 in 4 commonly used breast cancer and 3 prostate cancer cell lines. No mutations in exons 3, 4, 5, 6, 10, 11, and 12 of FOXP3 were detected in any of the cell lines examined (Figure 1A). To determine whether FOXP3 mutations occurred in the remaining exons, we also sequenced exons 2, 7, 8, and 9 in 34 of the 54 melanoma cell lines as well as in breast and prostate cancer cell lines. As for the previous analysis, no mutations in exons 2, 7, 8, and 9 were identified in any of the cell lines (Figure 1B). Exon 1 does not contain any coding sequence and was therefore not analyzed.

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Figure 1.  Representative chromatograms of (A) FOXP3 exon 11 and (B) FOXP3 exon 7 in PBMCs, melanoma, breast and prostate cancer cell lines. Red arrows indicate location of mutations previously found in breast and prostate tumors. PBMC, peripheral blood mononuclear cells. (C) BRAF exon 15 in melanoma cell lines with respective mutations (arrows).

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To validate that our melanoma cell line panel accurately represents primary metastatic melanoma, we determined the frequency of BRAF V600E/K mutations in this cell line panel, which has previously been reported to range between 40 and 60% in primary melanomas (Curtin et al., 2005). Mutations in BRAF (V600E or V600K) were identified in 35 of the 54 melanoma cell lines screened (65%) (Figure 1C), indicating that the BRAF mutation frequency of our cell line panel is comparable to that previously reported.

In summary, these findings indicate that in contrast to breast and prostate cancer, FOXP3 is not mutated in melanoma. Consistent with these findings, the mutational status of FOXP3 was also recently assessed in fresh bone marrow aspirates from 45 patients with acute myelogenous and lymphoblastic leukemias with no mutations observed (Kim et al., 2011a). Therefore, the somatic mutations and deletions in FOXP3 identified so far remain confined to tumors of epithelial origin, namely the breast and prostate, suggesting that its tumor suppressive function may be related to specific features of epithelial cell biology.

As discussed above, FOXP3 mutations in prostate cancer were identified by Wang et al., but were not observed in a cohort of Korean patients with prostate cancer (Kim et al., 2011b). Consistent with the findings of Kim et al., our analysis of 3 prostate cancer cell lines failed to identify any mutations in FOXP3. While the differences in FOXP3 mutation between the Wang and Kim studies may be due to the differences in the ethnic populations studied, additional studies are required to resolve this discrepancy.

Acknowledgement

  1. Top of page
  2. Acknowledgement
  3. References
  4. Supporting Information

BST is supported by Melbourne Research Scholarship, University of Melbourne. JM is an Australian Research Council Future Fellow. WC is an NHMRC Senior Research Fellow. MA is supported by Cancer Council Victoria. JC is supported by an Australian National Health and Medical Research Council Practitioner Fellowship.

References

  1. Top of page
  2. Acknowledgement
  3. References
  4. Supporting Information
  • Brunkow, M.E., Jeffery, E.W., Hjerrild, K.A., Paeper, B., Clark, L.B., Yasayko, S.A., Wilkinson, J.E., Galas, D., Ziegler, S.F., and Ramsdell, F. (2001). Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 27, 6873.
  • Curtin, J.A., Fridlyand, J., Kageshita, T. et al. (2005). Distinct sets of genetic alterations in melanoma. N. Engl. J. Med. 353, 21352147.
  • Ebert, L.M., Tan, B.S., Browning, J. et al. (2008). The regulatory t cell-associated transcription factor foxp3 is expressed by tumor cells. Cancer Res. 68, 30013009.
  • Gavin, M.A., Rasmussen, J.P., Fontenot, J.D., Vasta, V., Manganiello, V.C., Beavo, J.A., and Rudensky, A.Y. (2007). Foxp3-dependent programme of regulatory t-cell differentiation. Nature 445, 771775.
  • Hinz, S., Pagerols-Raluy, L., Oberg, H.H. et al. (2007). Foxp3 expression in pancreatic carcinoma cells as a novel mechanism of immune evasion in cancer. Cancer Res. 67, 83448350.
  • Kim, M.S., Chung, N.G., Yoo, N.J., and Lee, S.H. (2011a). No mutation in the foxp3 gene in acute leukemias. Leuk. Res. 35, e10.
  • Kim, M.S., Yoo, N.J., and Lee, S.H. (2011b). Absence of tumour suppressor foxp3 gene mutation in prostate cancers of korean patients. Pathology 43, 287.
  • Quaglino, P., Osella-Abate, S., Marenco, F., Nardo, T., Gado, C., Novelli, M., Savoia, P., and Bernengo, M.G. (2011). Foxp3 expression on melanoma cells is related to early visceral spreading in melanoma patients treated by electrochemotherapy. Pigment Cell Melanoma Res. 24, 734736.
  • Wang, L., Liu, R., Li, W. et al. (2009). Somatic single hits inactivate the x-linked tumor suppressor foxp3 in the prostate. Cancer Cell 16, 336346.
  • Wang, W.H., Jiang, C.L., Yan, W. et al. (2010). Foxp3 expression and clinical characteristics of hepatocellular carcinoma. World J. Gastroenterol. 16, 55025509.
  • Zhang, H.Y., and Sun, H. (2010). Up-regulation of foxp3 inhibits cell proliferation, migration and invasion in epithelial ovarian cancer. Cancer Lett. 287, 9197.
  • Zuo, T., Wang, L., Morrison, C. et al. (2007). Foxp3 is an x-linked breast cancer suppressor gene and an important repressor of the her-2/erbb2 oncogene. Cell 129, 12751286.

Supporting Information

  1. Top of page
  2. Acknowledgement
  3. References
  4. Supporting Information

Table S1. List of primers used for FOXP3 and BRAF sequencing.

Table S2. Clinical characteristics, staging, gender and BRAF mutational status of the melanoma, prostate and breast cancer cell lines.

FilenameFormatSizeDescription
PCMR_993_sm_TableS1-S2.doc152KSupporting info item

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