pISSN 1598-2998, eISSN 2005-9256 Cancer Res Treat. 2020;52(4):1273-1282 https://doi.org/10.4143/crt.2020.032 Original Article Open Access Forkhead Box C1 (FOXC1) Expression in Stromal Cells within the Microenvironment of T and NK Cell Lymphomas: Association with Tumor Dormancy and Activation Ji Hae Nahm, MD, PhD Purpose Woo Ick Yang, MD, PhD Forkhead box C1 (FOXC1) is critical for maintaining bone marrow microenvironments dur- Sun Och Yoon, MD, PhD ing hematopoiesis, but its role in hematological malignancies remains obscure. Here, we investigated whether FOXC1 regulates tumor dormancy and activation in the microenviron- ments of T and natural killer (NK) cell lymphomas. Materials and Methods One hundred and twenty cases of T and NK cell lymphomas were included; the immu- nohistochemical expression of FOXC1 was investigated in stromal cells, and numbers of FOXC1+ stromal cells were counted. Furthermore, the expression of phosphorylated p38 Department of Pathology, (p-p38) and phosphorylated ERK1/2 (p-ERK1/2) in tumor cells was investigated using Severance Hospital, Yonsei University immunohistochemistry. College of Medicine, Seoul, Korea Results FOXC1 was variably expressed in C-X-C motif chemokine 12–associated reticular stromal cells, histiocytes, (myo)fibroblasts, and endothelial cells. The phenotypes of cases were categorized as dormant (high p-p38/low p-ERK1/2; n=30, 25.0%), active (high p-ERK1/2/ low p-p38; n=25, 20.8%), or intermediate (others; n=65, 54.2%). Lower FOXC1+ stromal cell infiltration was associated with the dormant phenotype, the precursor T lymphoblastic + + + + + + + + + + + + + + + + + + + + + leukemia/lymphoma subtype, and inferior overall survival rates, whereas higher FOXC1 Correspondence: Sun Och Yoon, MD, PhD stromal cell infiltration was associated with the active phenotype and favorable patient + Department + + + + + of + Pathology, + + + + Severance+ + + + +Hospital, + + + + + + + + + + + + + + + + + + + + + + + + prognosis (p < 0.05 for all). + Yonsei + + + University + + + + College+ + + of+ Medicine,+ + + + + + + + + 50-1 + + Yonsei-ro, + + + + Seodaemun-gu, + + + + + + + + + + + + + Conclusion Seoul 03722, Korea + + + + + + + + + + + + + + + + + + + + These results suggested that FOXC1+ stromal cells within the microenvironments of T and + Tel: + + 82-2-2228-1763 + + + + + + + + + + + + + + + + + + Fax: + + 82-2-362-0860 + + + + + + + + + + + + + + + + + NK cell lymphomas might be related to tumor phenotypes. + E-mail: + + + [email protected] + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Key words + Received + + + + January + + + 13,+ + 2020 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + FOXC1, T and NK cell lymphomas, Tumor microenvironment, Stromal cells, Dormancy, Accepted July 3, 2020 p38, ERK + Published + + + + Online+ + + July+ + 3, + 2020 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Introduction tiation [6]. FOXC1 expression has been studied in epithelial tumor cells, such as those of nasopharyngeal carcinoma, The tumor microenvironment plays an important role in breast cancer, prostate cancer, and melanoma, and it plays tumorigenesis and tumor progression by fostering a cross- a regulatory role in the biological behavior of these tumors talk among tumor cells and several types of stromal cells via [7,8]. A recent study revealed that FOXC1 is preferentially various signaling pathways [1]. Recent studies have shown expressed in C-X-C motif chemokine 12 (CXCL12)–associated that the tumor microenvironment is involved in regulating reticular cells (CAR cells), and might promote CAR cell deve- tumor dormancy and activation [2,3]. FOX genes, encoding lopment, upregulating CXCL12 and stem cell factor expres- transcription factors of a family characterized by the presence sion [9]. CAR cells play essential roles in the tumor microenvi- of a forkhead box (Fox) DNA-binding domain, play key roles ronment and are involved in the regulation of hematopoietic in developmental processes during embryogenesis and tis- stem cells and disseminated tumor cells within the bone mar- sue differentiation [4,5]. The gene encoding forkhead box C1 row, affecting retention in the bone marrow, quiescence, and (FOXC1), located at 6p25, is involved in the pathogenesis of repopulation [10,11]. This connection between CAR cells and Hodgkin lymphoma, via the deregulation of B-cell differen- FOXC1 indicates that FOXC1 expression in the tumor micro- │ https://www.e-crt.org │ Copyright ⓒ 2020­­­ by the Korean Cancer Association 1273 This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Cancer Res Treat. 2020;52(4):1273-1282 environment is critical and specific for hematopoietic stem stage, International Prognostic Index (IPI) risk score, lactate cell niche formation and maintenance [12]. Although FOXC1 dehydrogenase (LDH) level, bone marrow involvement, has a potential role in stromal cells in regulating cell dor- and follow-up data were obtained from electronic medical mancy and activation within the tumor microenvironment, records, and prognostic implications were analyzed for 120 similar to CAR cells, studies on FOXC1 expression in stro- patients with available clinical data. The mean follow-up mal cells within tumor microenvironments of hematological period after diagnosis was 14 months (range, 1 to 184 malignancies are limited. months). The clinicopathological features of the patients are The imbalance in activation of the p38 mitogen-activated summarized in S1 Table. protein kinase (MAPK) pathways and extracellular signal regulated kinase (ERK) MAPK pathways plays a key role 2. TMA construction and immunohistochemical evalua- in the establishment of tumor dormancy and activation. In tion dormant tumors, phosphorylated p38 MAPK is generally TMA construction and immunohistochemical staining of more active than phosphorylated ERK MAPK, and vice versa TMA blocks were performed following a standard protocol with respect to tumor activation [2,3]. Studies also show that using a Ventana automatic immunostainer (Ventana, Bench- the p38 and ERK MAPK pathways are involved in normal mark, Tuscan, AZ) as previously described [18]. The follow- immature T cell differentiation. Increased p38 MAPK activa- ing primary antibodies were used: FOXC1 (1:50, Abcam, tion blocks T cell differentiation, whereas increased ERK1/ Cambridge, UK), CXCL12 (1:50, CXCL12/SDF1, R&D Sys- ERK2 activation is required for T cell differentiation [13,14]. tems, Minneapolis, MN), phosphorylated p38 (p-p38; 1:100, Here, we hypothesized that FOXC1 might be involved in phospho T180+Y182, Abcam), phosphorylated ERK1 and regulating tumor dormancy and activation within the tumor ERK2 (p-ERK1/2; 1:100, Erk1 [pT202/pY204]+Erk2 [pT185/ microenvironments of T cell and natural killer (NK) cell lym- pY187], Abcam), CD163 (1:100, clone MRQ-26, Cell Marque, phomas, which occur frequently at various extramedullary Rocklin, CA), CD68 (1:150, clone PG-M1, Dako, Glostrup, sites, disseminate regularly into the bone marrow, and are Denmark), CD34 (1:50, clone QBEnd 10, Dako), S100 (1:2,000, clinically aggressive [15]. In the present study, we investigated Dako), and α-smooth muscle actin (1:500, clone 1A4, Dako). the implications of FOXC1 expression in the tumor microen- To identify FOXC1+ and CXCL12+ stromal cells, the number vironments of T and NK cell lymphomas, focusing on tumor of spindled reticular cells with positive immunoexpression dormancy and activation with respect to the p38 MAPK and in the cytoplasm was counted in at least five fields of the cap- ERK MAPK pathways. tured microscopic photographs at 400× magnification and the average cell number was obtained. The cutoff for high FOXC1+ stromal cell infiltration was defined as the number Materials and Methods of FOXC1+ stromal cells that surpassed the median value of the 120 tested cases. To evaluate p-p38 and p-ERK1/2 immu- 1. Case selection and clinicopathological analysis noexpression in tumor cells, moderate to strongly stained In total, 120 cases of T and NK cell lymphomas diagnosed nuclei of tumor cells were counted and divided by the total at the Severance Hospital from 1999 to 2013 were selected. cell count of the same field. These markers were also counted Consecutive patients with pathological diagnoses of T and in at least five high-power fields using photographs and the NK cell lymphomas based on biopsied or excised specimens average proportion was obtained. Protein expression was were included in this study. Patients with remaining tissue divided into high and low using median values. inadequate to construct tissue microarrays (TMAs) or per- form immunohistochemical staining were excluded. The 3. Genetic analyses using acute myeloid leukemia cases included subtypes were 35 cases of peripheral T cell lym- based on the Cancer Genome Atlas Network data phoma, not otherwise specified (PTCL, NOS), 39 cases of The mRNA expression levels of FOXC1, p38 MAPKs (MAP- extranodal natural killer/T cell lymphoma (NKTL), 12 cases K11, MAPK14, MAPK12, and MAPK13), ERK MAPKs (MAP- of angioimmunoblastic T cell lymphoma (AITL), 20 cases of K1/ERK2 and MAPK3/ERK1), and DUSP1 (MAPK phos- anaplastic large-cell lymphoma (ALCL), anaplastic lympho- phatase 1, MKP-1; the regulator of MAPK phosphorylation) ma kinase (ALK)-positive,