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FMNL2/FMNL3 Formins Are Linked with Oncogenic Pathways and Predict Melanoma Outcome

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FMNL2/FMNL3 Formins Are Linked with Oncogenic Pathways and Predict Melanoma Outcome The Journal of Pathology: Clinical Research J Path: Clin Res January 2016; 2: 41–52 Original Article Published online 10 December 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/cjp2.34 FMNL2/FMNL3 formins are linked with oncogenic pathways and predict melanoma outcome Maria Gardberg,1* Vanina D Heuser,1 Ilkka Koskivuo,2 Mari Koivisto3 and Olli Carpen1,4 1 Department of Pathology, University of Turku and Turku University Hospital, Turku, Finland 2 Department of Plastic and General Surgery, Turku University Hospital, Turku, Finland 3 Department of Biostatistics, University of Turku, Turku, Finland 4 Auria Biobank, Turku University Hospital, Turku, Finland *Correspondence to: Maria Gardberg, Department of Pathology,Turku University Hospital, Kiinamyllynkatu 10, 20520 Turku, Finland. e-mail: [email protected] Abstract While most early (stage I-II) melanomas are cured by surgery, recurrence is not uncommon. Prognostication by current clinicopathological parameters does not provide sufficient means for identifying patients who are at risk of developing metastases and in need of adjuvant therapy. Actin-regulating formins may account for inva- sive properties of cancer cells, including melanoma. Here, we studied formin-like protein 2 and 3 (FMNL2 and FMNL3) in melanoma by analysing their role in the invasive properties of melanoma cells and by evaluating whether FMNL2 expression is associated with melanoma outcome. Immunohistochemical characterization of FMNL2 in a cohort of 175 primary cutaneous stage I-II melanomas indicated that high FMNL2 reactivity corre- lates with poor outcome as evaluated by recurrence free survival (p < 0.0001) or disease specific survival (p < 0.0001). In multivariate analysis, Breslow’s thickness (p < 0.05) and FMNL2 expression (p < 0.001) remained as independent prognostic factors. Cellular studies revealed that FMNL2 is a component of filopodia in many melanoma cell lines. Inhibition of either FMNL2 or the closely related FMNL3 affected the mainte- nance of melanoma cell morphology and reduced migration. Finally, inhibition of the BRAF, PI3K and MAPK oncogenic pathways markedly reduced expression of both FMNL2 and FMNL3 in melanoma cells. The results suggest a major role for FMNL2/FMNL3 formins in melanoma biology and raise the possibility that the novel targeted melanoma drugs may interfere with the cellular properties regulated by these formins. Keywords: melanoma; formin; actin; FMNL2; FMNL3 Received 25 August 2015; accepted 2 December 2015 All authors declare that they have no conflict of interest. Contract/grant details: This study was supported by funding from the Academy of Finland, Finska L€akares€allskapet, the Sigrid Juselius Foundation, the K Albin Johansson Foundation, the Perklen Foundation, the Finnish Medical Society Duodecim, the Finnish Cancer Organizations, and Turku University Hospital Research Funds. MG has during this study been supported by the National School of Clinical Investigation. Introduction tant metastases, the prognosis is dismal, the 5-year sur- The incidence of cutaneous melanoma is rapidly rising vival rate being 15–20% (www.cancer.org). with 230 000 new cases and 55 000 cancer deaths annu- The methods for identifying individuals with ally (WHO International Agency of Cancer Research, adverse prognosis are currently inaccurate and lack globocan.iarc.fr). Melanomas are staged clinically any molecular parameters. The evaluation relies on based on the level of dissemination: stages I and II clinical features such as age, gender and location of being localized, stage III having lymph node metastases primary tumour, as well as histological findings. In and stage IV having distant metastases. The treatment localized melanoma, these are tumour thickness, of localized melanoma is surgical, which in most cases presence of ulceration, mitotic count and the presence is curative. However, melanoma recurrence is not of tumour-infiltrating lymphocytes (TILs) [1,2]. uncommon, sometimes after a decade-long remission. There is great need for biomarkers that could help to Recurrence may present locally but more commonly as identify the patients at risk of metastatic progression, lymph node metastasis or distant metastasis. With dis- as such patients might benefit from adjuvant therapy. VC 2015 The Authors The Journal of Pathology: Clinical Research published by J Path: Clin Res April 2016; 2: 41–52 The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. 42 M Gardberg et al Identification of over-active signalling pathways in Methods melanoma has guided development of targeted thera- pies for metastatic disease. The pivotal signalling path- Melanoma specimens ways active in melanoma are the Ras/Raf/MEK/ERK The melanoma cohort consists of 175 consecutive [also known as the MAPK (mitogen activated protein archive tissue samples and follow-up data from kinase)] and the phosphoinositide-3-OH kinase (PI3K) patients with primary invasive cutaneous melanoma signalling pathways. Abnormal activity of these path- of the American Joint Committee on Cancer (AJCC) ways, induced by oncogenic mutations, leads to clinical stages I and II, operated at the Turku Univer- increased proliferation and cancer cell survival [3]. sity Hospital in 1990–2005. Clinical stages I and II The most common mutation, present in more than 50% together represent localized melanomas of any thick- of melanomas, is BRAF V600, which leads to constitu- ness without clinical nodal involvement or distant tive activation of ERK [4,5]. When present, BRAF metastases [1]. At Turku University Hospital, sentinel inhibitors such as vemurafenib improve survival of node biopsy was initiated in 2001. The cases with patients with metastatic disease. The effect of BRAF detected micrometastases in sentinel nodes were inhibition is unfortunately lost by acquired resistance, included (postoperative AJCC stage III with nodal which typically develops within 6 months [6]. involvement, n 5 11). If a metastasis was histologi- Formins are an actin nucleating protein family cally sampled during follow-up, it was also included. with diverse actin-regulating and potentially pro- This amounted to 34 metastasis samples. The clinico- invasive functions [7]. Among the sub-family of pathological parameters relevant for prognostication diaphanous-related formins are two members, formin- of melanoma (ie age, gender, anatomical location, like protein 2 (FMNL2 also known as FRL3) and Breslow thickness, Clark level, ulceration, mitosis formin-like protein 3 (FMNL3 also known as FRL2), count, TILs) were re-evaluated for this study by at which share extensive sequence homology. FMNL2 least two pathologists. All patients underwent wide and FMNL3 have both actin polymerizing and bun- local excision with histologically confirmed tumour- dling capacity [8,9]. The activity of these formins free margins. Suspected metastases were further appears to be interconnected as FMNL2 and FMNL3 investigated and mostly treated by surgery, chemo- have been shown to form heterodimers [9]. When therapy, interferon or radiotherapy. The patients were transfected with cDNA constructs coding for consti- followed up at the Department of Oncology and tutively active FMNL2 or FMNL3 forms, the proteins Radiotherapy at Turku University Hospital. A recur- are targeted to filopodia in mouse melanoma and rence was recorded when a new local tumour, in- human T-cell lymphoma cell lines. Previous in vitro transit metastasis or nodal/distal metastasis appeared. studies have suggested that FMNL2 partakes in mela- The most recent follow-up information on the noma cell invasion [10,11], whereas no information patients was updated from electronic medical records on FMNL3 exists in this respect. Our earlier studies in June 2012. The final follow-up date of each have shown that FMNL2 is widely expressed in patient was defined as the date of the most recent human tissues, and found both in skin keratinocytes hospital call or the date of death. The cause and time and cultured melanoma cells [12]. Due to the lack of of death ware obtained from patient records, autopsy suitable FMNL3 antibodies, its expression in human reports or from the Statistics Finland’s Archive of tissues has not been characterized. Death Certificates. In the present study, we have characterized the role and The Joint Committee on Ethics of the University interplay of FMNL2/FMNL3 formins in melanoma, both of Turku and Turku University Hospital approved the by studying a melanoma cohort with extensive follow-up use of the tissue collection for this study. According and by in vitro methods. By immunohistochemical evalua- to Finnish legislation, archived tissue samples col- tion, we show that the level of FMNL2 expression is a lected for diagnostic purposes may be used with the strong independent indicator of recurrence-free and permission of the local ethical authority. melanoma-specific survival in primarily localized disease. At the cellular level, we show that endogenous FMNL2 and FMNL3 are filopodial components in melanoma cell Immunohistochemistry lines, and show that depletion of FMNL2 and/or FMNL3 The paraffin-embedded specimens were stained with leads to altered cell morphology and decreased migration in a rabbit anti-human FMNL2 polyclonal
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