Ectopic expression of embryonic stem cell and other developmental genes in cutaneous T-cell lymphoma

Abstract

Cutaneous T-cell lymphoma (CTCL) is a potentially devastating malignancy. The pathogenesis of this cancer remains poorly elucidated. Previous studies focused on analysis of expression and function of known oncogenes and tumor suppressor genes. However, emerging reports highlight that it is also important to analyze the expression of genes that are ectopically expressed in CTCL (e.g., embryonic stem cell genes (ESC), cancer testis (CT) genes, etc.). Currently, it is not known whether ESC genes are expressed in CTCL. In the current work, we analyze by RT-PCR the expression of 26 ESC genes, many of which are known to regulate pluripotency and promote cancer stem cell-like phenotype, in a historic cohort of 60 patients from Boston and in a panel of 11 patient-derived CTCL cell lines and compare such expression to benign inflammatory dermatoses that often clinically mimic CTCL. Our findings document that many critical ESC genes including NANOG, SOX2, OCT4 (POU5F1) and their upstream and downstream signaling members are expressed in CTCL. Similarly, polycomb repressive complex 2 (PRC2) genes (i.e., EZH2, EED, and SUZ12) are also expressed in CTCL lesional skin. Furthermore, select ESC genes (OCT4, EED, TCF3, THAP11, CHD7, TIP60, TRIM28) are preferentially expressed in CTCL samples when compared to benign skin biopsies. Our work suggests that ESC genes are ectopically expressed together with CT genes, thymocyte development genes and B cell-specific genes and may be working in concert to promote tumorigenesis. Specifically, while ESC genes may be promoting cancer stem cell-like phenotype, CT genes may be contributing to aneuploidy and genomic instability by producing aberrant chromosomal translocations. Further analysis of ESC expression and function in this cancer will greatly enhance our fundamental understanding of CTCL and will help us identify novel therapeutic targets.

Introduction

CTCL is a rare, but potentially devastating malignancy. It represents a heterogeneous group of non-Hodgkin's lymphomas that are characterized by localization of malignant T lymphocytes to the skin.¹ The annual incidence of CTCL was recently reported to be ∼10 cases per million individuals per year in the United States. Mycosis fungoides (MF), Anaplastic large cell lymphoma (ALCL), and Sézary syndrome (SS) represent some of the most common forms of CTCL and together account for >80% of all CTCL cases.¹

The molecular pathogenesis of CTCL remains only partially understood. Recent reports elucidated the nature of cancer initiating cells for MF and SS.² Multiple studies attempted to clarify the genetic multistep carcinogenesis of CTCL.^3-6 In addition, several studies identified recurrent chromosomal aberrations in MF/SS.^7-12 While much research is focused on investigation of known oncogenes and tumor suppressor genes in CTCL,^6,13-15 recent reports suggest that it is also important to look for genes that could be erroneously expressed in this cancer.¹⁶ Ectopic expression of oncodevelopmental genes (e.g., α-Fetoprotein, H19 and Sonic Hedgehog signaling genes), ESC genes (e.g., OCT4 (POU5F1), SOX2, NANOG, EED, SUZ12, etc.), and CT genes have been reported in various solid and lymphoproliferative malignancies, where they are believed to play a central role in tumorigenesis and cancer progression. The expression status of these genes in CTCL remains unknown.

Recent reports demonstrated that CT genes are ectopically expressed in this malignancy. We and others have suggested that CT antigens during carcinogenesis may play an important role in maintaining cell survival (i.e., inhibition of apoptosis),^17-19 promoting resistance to various forms of chemo- and radio-therapy^20,21 and contributing to oncogenesis by targeting p53 and p21 tumor suppressor genes.^22,23 Also, since SYCP1 (Synaptonemal Complex Protein 1), SYCP3, DMC1 (Disrupted Meiotic cDNA 1), and REC8 CT antigens under normal conditions regulate generation of double strand DNA breaks during crossing over in meiosis, it was suggested that these genes may promote aneuploidy and genomic instability in cancers by producing aberrant chromosomal translocations.²⁴

Most importantly, in recent years a concept of cancer stem cells (CSC) has emerged, where these cells share many characteristics with normal stem cells, specifically, they possess an enhanced self-renewal capacity, resistance to apoptosis, ability to maintain undifferentiated state, overcome cellular senescence and give rise to all cell types found in a particular tumor. Like normal stem cells, CSCs divide infrequently and, therefore, are often spared by therapies that target rapidly dividing populations of cells. These CSCs are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Hence, understanding the expression of these embryonic genes in CTCL may help us better understand the pathogenesis of this cancer and identify novel diagnostic markers and therapeutic targets.

In the current work, we investigate the expression of a panel of ESC genes in CTCL lesional skin and in 11 patient-derived CTCL cell lines and summarize expression patterns for CT antigens, thymocyte development, B cell-specific, and other ectopically expressed genes in this cancer.

Results

Characterization and function of CSCs has recently become the focus of cancer research. While expression of many ESC and putative CSC markers has been extensively studied in other cancers, very little experimental data are available for CTCL. To address this, we tested the expression of 26 ESC markers in CTCL lesional skin samples via RT-PCR. These results demonstrate that, while few genes are not expressed or infrequently expressed in CTCL (e.g., SALL4, ESRRB and DAX1/NR0B1), the majority of ESC markers are heterogeneously expressed in CTCL (Fig. 1A and Table S1). Furthermore, a number of genes that are critical for maintenance of pluripotency in ESCs (e.g., EED, SUZ12, EZH2, MTF2, OCT4, CHD7, and TIP60) are almost universally expressed in CTCL lesional skin samples. Normal function of these genes and their potential roles in carcinogenesis are summarized in Table S1.

Figure 1.

Expression of embryonic stem cell (ESC) genes in CTCL. (A) Lesional skin biopsies were obtained from CTCL patients and gene expression for a panel of ESC genes was analyzed via RT-PCR. (B) Similarly, the expression of ESC genes was also tested by RT-PCR in 11 patient-derived CTCL cell lines cultured under standard conditions.

Any given CTCL lesional skin biopsy contains numerous cell types including keratinocytes, fibroblasts, merkel cells, melanocytes as well as infiltrating malignant, and reactive CD4⁺ T cells. To confirm that the above observed ectopic expression of ESC genes takes place in malignant T cells we tested the expression of these 26 ESC genes in a panel of 11 patient-derived immortalized CTCL cell lines (Fig. 1B). Our findings confirm that many of these genes are also ectopically expressed in patient-derived CTCL cell lines.

We also analyzed the expression of genes that are usually not expressed in mature CD4⁺ T cells. TOX (Thymocyte selection-associated high mobility group box) and EVA1 (Epithelial C-like antigen 1) are usually expressed in thymocyte development, but are subsequently downregulated in mature T cells,^25,26 while BLK (B-lymphoid kinase) and POU2AF are usually specific to B cells and should not be expressed in T cells.^27,28 PLS3 (Plastin 3) is an actin binding protein, which is also normally not expressed in T cells.^6,29 Our RT-PCR analysis confirms that BLK, POU2AF, TOX, EVA1, and PLS3 are expressed in CTCL lesional skin biopsies (Fig. 2).

Figure 2.

Expression of selected developmental genes in CTCL. RT-PCR expression of thymocyte development genes (EVA1 and TOX), B cell-specific genes (BLK and POU2AF) and PLS3 in lesional skin biopsies from CTCL patients.

We then compared the expression of these genes between CTCL lesional skin samples, benign dermatoses that often clinically masquerade as CTCL (e.g., chronic eczema, psoriasis, and pityriasis rubra pilaris) and normal skin samples from healthy volunteers. Our findings indicate that select genes (OCT4, EED, TCF3, THAP11, CHD7, TIP60, TRIM28, and BLK) are preferentially expressed in CTCL samples when compared to benign skin biopsies (Fig. 3).

Figure 3.

Comparison by RT-PCR of gene expression for ESC and developmental genes between normal skin, skin affected by benign inflammatory dermatoses (i.e., psoriasis, chronic eczema and pityriasis rubra pilaris or PRP) vs. lesional skin from CTCL patients. OCT4, EED, TCF3, THAP11, CHD7, TIP60, TRIM28, and BLK genes are preferentially expressed on mRNA level in CTCL samples when compared to benign skin conditions.

Discussion

Our results for the first time document the expression of ESC genes in CTCL lesional skin biopsies and patient-derived cell lines. Furthermore, we demondtrate that OCT4, EED, TCF3, THAP11, CHD7, TIP60, and TRIM28 ESC genes are preferentially expressed in CTCL, but not in benign inflammatory dermatoses. From the extensive list of ESC genes (Table S1) few classes especially stand out. PRC2 genes (EZH2, EED, and SUZ12) were previously shown to promote pluripotency in normal stem cells, enhance self-renewal capacity, maintain de-differentiation, and resist apoptosis in normal ESCs.^30-32 A schematic diagram of the role of these genes in carcinogenesis is presented in Fig. 4. Polycomb group proteins are key regulators of chromatin structure, cell identity, and development.³⁰ Indeed, it was suggested that reprogramming of somatic cells toward pluripotency would involve extensive chromatin reorganization and changes in gene expression. These genes were documented to be ectopically expressed in multiple malignancies, where they establish/promote a cancer stem cell-like state.^30-32 Such ectopic expression of PRC2 components often correlates with disease severity.^30,32 As highlighted in in our study, EZH2, EED and SUZ12 are strongly expressed in CTCL samples, while EED is preferentially expressed in CTCL, but not in benign dermatoses that often clinically mimic this disease. Similarly, our study indicates that JARID2, protein that often associates with the PRC2 complex,³³ as well as PHC1 member of the PRC1-like complex,^34,35 are also ectopically expressed in CTCL lesional skin. JARID2 is heterogeneously expressed in cell lines derived from SS patients and in MF patient biopsies. All three genes were shown to be important in maintaining ESC identity ( Table S1).

Figure 4.

Putative function of PRC2 complex genes in carcinogenesis. Integrity of PRC1 and PRC2 signaling is critical for embryonic and adult stem cells. Ectopic expression of PRC1 and PRC2 in cancer may reprogram somatic cells toward cancer stem cell phenotype. Deregulation of these genes in somatic adult tissues may play a critical role in tumorigenesis.

NANOG, SOX2, and OCT4 are another group of key transcription factors involved in the maintenance of pluripotency of ESCs.^36-38 These genes are essential for maintaining the self-renewing undifferentiated state of the inner cell mass of the blastocyst.³⁹ OCT4, SOX2, and NANOG are ectopically expressed in many malignancies, where they are often associated with aggressive disease and poor cancer survival.^40-46 Similarly to EZH2, EED, and SUZ12, these genes work in concert to induce pluripotent ESC-like state and promote cancer stem cell phenotype.^47,48 Based on our RT-PCR expression results these genes are heterogeneously expressed in CTCL lesional skin, while OCT4 is preferentially expressed in CTCL lesional skin, but not in benign skin conditions. Furthermore, consistent with the above findings, upstream and downstream members of NANOG signaling are also expressed in CTCL. ZFX (Zinc finger protein X-linked), a transcription factor, is known to transactivate the promoters of NANOG and SOX2,^49,50 while DAX1 (also known as NR0B1) and MTF2 pluripotency markers are induced by NANOG.³⁹ All of the aforementioned genes are heterogeneously expressed in CTCL lesional skin. Overexpression of ZFX contributes to the ‘stemness’ and pluripotent behavior of cancers.^49,50 Our study further demonstrates that CTCL lesional skin and patient-derived cell lines express other pluripotency ESC markers including CNOT3, KLF4, TBX3, and TRRAP. Detailed description of these genes and their biological activities is provided in Table S1. In contrast, few ESC genes (e.g., SALL4 and ESRRB) were not expressed in CTCL lesional biopsies.

Looking at the other classes of genes, we and others have previously documented that several CT genes were ectopically expressed in CTCL.¹⁶ Normal function of these genes is to regulate meiosis, synaptonemal complex assembly, and generation of double strand DNA breaks during crossing over.¹⁶ Ectopic expression of these genes in cancer may promote genomic instability and be an important driving force behind aneuploidy and generation of balanced and unbalanced chromosomal translocations. Hence, while ESC genes may be reprogramming cancer stem cell-like phenotype, CT genes may be promoting genomic instability enabling these cells to express important oncogenes and disrupt critical tumor suppressor genes.

Finally, recent studies highlighted that malignant T cells in CTCL are able to express few B cell-specific genes one of which is the Src kinase BLK.²⁷ Importantly, BLK is constitutively active in malignant T cells and appears to be a bona fide oncogene which drives malignant T cell proliferation in vitro and tumor formation in vivo.^27,51 In addition, recent translational experimental work revealed that TOX expression, which is usually silenced in mature T cells, can be used as a robust prognostic and diagnostic marker for MF and SS,^52,53 while PLS3 actin binding protein, which is usually not expressed in T cells is consistently expressed in CTCL.^6,29,54,55 A growing body of literature documents that ectopic expression of these genes in CTCL is not a mere indication of deregulated cellular processes, but an important mechanism of tumorigenesis and cancer progression. Our study confirms that select thymocyte development genes (e.g., EVA1 and TOX), B cell-specific genes (POU2AF and BLK) and PLS3 are expressed in CTCL lesional skin and in patient-derived cell lines. A summary of different classes of ectopically expressed genes in this cancer is presented in Fig. 5.

Figure 5.

Summary of different classes of aberrantly expressed genes in CTCL. Aberrant expression of embryonic stem cells genes, cancer testis genes, B cell-specific genes and thymocyte development genes may play a role in tumorigenesis/cancer progression in CTCL. Specifically, while embryonic stem cell genes (e.g., members of the PRC2 complex) may be reprogramming cancer stem cell phenotype, cancer testis genes may be contributing to aneuploidy and genomic instability by producing aberrant chromosomal translocations.

In conclusion, our work highlights the importance of ectopic expression of ESC genes, CT antigens, B cell-specific, and thymocyte development genes in CTCL. Further analysis of how ECS genes reprogram malignant T cells and promote cancer stem cell phenotype will greatly enhance our fundamental understanding of this cancer and will help us identify novel therapeutic targets.

Materials and Methods

Patients and samples

All patients were enrolled in the IRB-approved study protocol with informed consent in accordance with the Declaration of Helsinki.^3,4 CTCL patients were recruited from the Cutaneous Lymphoma Clinic at the Dana Farber Cancer Institute (DFCI)/Brigham and Women's Hospital (BWH). All tissue samples were obtained and processed as previously described.⁴ Briefly, punch biopsies from involved skin were collected from 60 CTCL patients between January 26, 2003 and June 1, 2005. The diagnosis and clinical staging were established according to the diagnostic criteria of CTCL.⁵⁶ The obtained 6 mm biopsies were immediately snap-frozen in liquid nitrogen. Tissue was powdered in liquid nitrogen using Cryo-Press (Microtec Co, Chiba, Japan), and total RNA was extracted using Trizol reagent (Invitrogen, Catalog # 15596-026) and converted to cDNA using the iScript cDNA synthesis kit (Bio-Rad, Catalog # 170-8896) according to the manufacturer's instructions. The historic cohort of patients from Boston (n = 60) that was initially reported in 2007,⁴ was at the heart of extensive research that led to multiple publications in the field.^{3,4,13,14,16,53,57,58} The biopsy samples analyzed in this report are the same samples that were analyzed in previous studies.^{3,4,13,14,16,57,58} Similarly, volunteers with normal healthy skin (N = 5) and benign inflammatory dermatoses (N = 19) were recruited from the outpatient dermatology clinic of the University of British Columbia (Vancouver, Canada) with informed consent.^18,20 Full-thickness lesional skin punch biopsies were obtained under local anesthesia as previously described.^14,53,57

Cell culture

HH, H9, Hut78, MJ and Hut102 patient-derived CTCL cell lines were previously described^59,60 and were purchased from the American Tissue Culture Collection (ATCC). H9 is a clonal derivative of Hut78 cell line.⁶¹ MyLa, PB2B, Mac2A, SZ4, SeAx, Sez4 were a generous gift from professors K. Kaltoft and N. Ødum (Copenhagen, Denmark) and were previously described elsewhere.^62-66 MJ, Hut78 cells were serially passaged in IMDM media (Invitrogen, Catalog # 12440-079) containing 10% fetal bovine serum (FBS) (Invitrogen, Catalog # 26140-079). HH, H9, Hut102, MyLa, Mac2A and SZ4 cells were grown in RPMI media (Invitrogen, Catalog # 11875-093) containing 10% FBS. Finally, Sez4 and SeAx cells were grown in RPMI media containing 10% FBS, 5 ng/mL of recombinant human IL-2 (Catalog # 202-IL-010 from R&D Systems) and IL-4 (Catalog # 204-IL-010 from R&D Systems). All cells were grown in 5% CO₂, 95% air humidified incubator at 37°C. Total RNA was extracted using Trizol reagent (Invitrogen) and converted to cDNA using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions.

Quantitative real-time reverse transcription-PCR gene expression analysis

Gene expression was tested via RT-PCR in CTCL patients’ lesional skin and in patient-derived cell lines as previously described.^3,13,14 Briefly, RT-PCR was performed utilizing the obtained cDNA from patients and cell lines using iScript RT-PCR mix (Bio-Rad, Catalog #170-8893) on Bio-Rad iCycler (Bio-Rad, Catalog # 185-5201). Primer pair sequences for tested genes and control housekeeping genes are listed in Table S2. The expression was standardized using genorm method⁶⁷ utilizing ACTB, B2M, SDHA, YWHAZ, and HMBS housekeeping genes.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the Canadian Dermatology Foundation research grants to Dr. Sasseville, Dr. Litvinov and Dr. Zhou, the Fonds de la recherche en santé du Québec (FRSQ) research grant to Dr. Sasseville, Canadian Institutes of Health Research to Dr. Zhou, and the National Institutes of Health SPORE program (P50 CA093683) to Dr. Kupper.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website.