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Cell Cycle Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kccy20
Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines ae
ae
be
b
a
Ivan V Litvinov , Brendan Cordeiro , Simon Fredholm , Niels Ødum , Hanieh Zargham , c
c
a
d
b
Yuanshen Huang , Youwen Zhou , Kevin Pehr , Thomas S Kupper , Anders Woetmann & Denis Sasseville a
a
Division of Dermatology; McGill University Health Centre; Montréal, QC Canada
b
Department of International Health; Immunology and Microbiology; University of Copenhagen; Copenhagen, Denmark c
Click for updates
Department of Dermatology and Skin Science; University of British Columbia; Vancouver, BC Canada d
Harvard Skin Disease Research Center; Department of Dermatology; Brigham and Women's Hospital; Harvard University; Boston, MA USA e
These authors have contributed equally to this work. Published online: 30 Oct 2014.
To cite this article: Ivan V Litvinov, Brendan Cordeiro, Simon Fredholm, Niels Ødum, Hanieh Zargham, Yuanshen Huang, Youwen Zhou, Kevin Pehr, Thomas S Kupper, Anders Woetmann & Denis Sasseville (2014) Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines, Cell Cycle, 13:18, 2975-2982, DOI: 10.4161/15384101.2014.947759 To link to this article: http://dx.doi.org/10.4161/15384101.2014.947759
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
REPORT Cell Cycle 13:18, 2975--2982; September 15, 2014; © 2014 Taylor and Francis Group, LLC
Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines Ivan V Litvinov1,y, Brendan Cordeiro1,y, Simon Fredholm2,y, Niels Ødum2, Hanieh Zargham1, Yuanshen Huang3, Youwen Zhou3, Kevin Pehr1, Thomas S Kupper4, Anders Woetmann2,*, and Denis Sasseville1,* 1
Division of Dermatology; McGill University Health Centre; Montreal, QC Canada; 2Department of International Health; Immunology and Microbiology; University of Copenhagen; Copenhagen, Denmark; 3Department of Dermatology and Skin Science; University of British Columbia; Vancouver, BC Canada; 4Harvard Skin Disease Research Center; Department of Dermatology; Brigham and Women’s Hospital; Harvard University; Boston, MA USA y
These authors have contributed equally to this work.
Downloaded by [Brandeis University] at 02:55 25 November 2014
Keywords: Cutaneous T-Cell Lymphoma (CTCL), Mycosis Fungoides (MF), mi-R155, Sezary Syndrome (SS), STAT6 and STAT4 Abbreviations: CTCL, cutaneous T-cell lymphoma; MF, mycosis fungoides; SS, sezary syndrome; HDAC, histone deacetylase; STAT, signal transducers and activators of transcription.
Deregulation of STAT signaling has been implicated in the pathogenesis for a variety of cancers, including CTCL. Recent reports indicate that loss of STAT4 expression is an important prognostic marker for CTCL progression and is associated with the acquisition of T helper 2 cell phenotype by malignant cells. However, little is known about the molecular mechanism behind the downregulation of STAT4 in this cancer. In the current work we test the expression of STAT4 and STAT6 via RT-PCR and/or Western Blot in CTCL lesional skin samples and in immortalized patient-derived cell lines. In these malignant cell lines we correlate the expression of STAT4 and STAT6 with the T helper (Th) phenotype markers and test the effect of Histone Deacetylase (HDAC) inhibitors and siRNA-mediated knock down of miR-155 on STAT4 expression. Our findings demonstrate that STAT4 expression correlates with Th1 phenotype, while STAT6 is associated with the Th2 phenotype. Our results further document that STAT4 and STAT6 genes are inversely regulated in CTCL. Treatment with HDAC inhibitors upregulates STAT4 expression, while at the same time decreases STAT6 expression in MyLa cells. Also, siRNA-mediated knock down of miR-155 leads to upregulation in STAT4 expression in MyLa cells. In summary, our results suggest that loss of STAT4 expression and associated switch to Th2 phenotype during Mycosis Fungoides progression may be driven via aberrant histone acetylation and/or upregulation of oncogenic miR-155 microRNA.
Introduction Cutaneous T-Cell Lymphoma (CTCL) is a rare, but potentially devastating malignancy. Mycosis Fungoides (MF) and its leukemic form, Sezary Syndrome (SS), are the most common variants and account for »50% of all cutaneous lymphomas.1 In Caucasians MF/SS primarily affects individuals over 55 years of age, while in African-Americans, Hispanics and Arabic individuals this disease presents at a significantly younger age (i.e. 20s and 30s).2-4 Multiple epidemiologic studies documented that the incidence of CTCL has increased by »3 fold in the last 25– 30 years.4,5 In the early disease stages, which can last for several years, MF presents as flat erythematous skin patches resembling benign inflammatory diseases, whereas in the later stages, MF cells gradually form plaques or tumors and may disseminate to the lymph
nodes and internal organs.1 The early stages of CTCL are often difficult to distinguish clinically and even histologically from other benign entities including chronic eczema, psoriasis and pityriasis rubra pilaris.6 In advanced disease, malignant cells may appear in the peripheral blood, leading to the leukemic stage of CTCL. A subset of leukemic CTCL, known as SS, is characterized by a triad of erythroderma, lymphadenopathy and detection of malignant T cells with convoluted/cerebriform nuclei on a peripheral blood smear.1 The life expectancy of SS patients is usually less than 3 years.1 The molecular pathogenesis of CTCL remains only partially understood. Recent reports elucidated the nature of cancer initiating cells for MF and SS.7 Multiple studies attempted to clarify the genetic multistep carcinogenesis of CTCL.8-11 Persistent activation of transcription factors of the signal transducers and activators of transcription (STAT) protein family has been
*Correspondence to: Denis Sasseville; Email: [email protected]; Anders Woetmann; Email: mailto:[email protected] Submitted: 05/16/2014; Revised: 07/01/2014; Accepted: 07/03/2014 http://dx.doi.org/10.4161/15384101.2014.947759
www.landesbioscience.com
Cell Cycle
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Downloaded by [Brandeis University] at 02:55 25 November 2014
implicated in the pathogenesis of a wide variety of human cancers, including CTCL. Expression and function of STAT3, STAT4 and STAT5 have been extensively studied in CTCL and these genes appear to play an important role in the disease pathogenesis and can be used as important prognostic markers. Upregulation of STAT5 signaling occurs in the early stages of CTCL.12,13 A growing body of experimental evidence suggests that this gene is important for expression of anti-apoptotic proteins (bcl-2 and bcl-x), cell cycle genes (Cyclin D and c-myc) and oncogenic miR-155 microRNA,14-16 which work together to promote cancerogenesis. miR-155 was dubbed as the “bridge between inflammation and cancer”.17 In the early stages of CTCL, STAT4 appears to be overexpressed, when compared to non-malignant skin samples.18 The expression of STAT4, which is required for T helper (Th) 1 differentiation, is subsequently lost at the later stages, where the disease acquires predominantly Th2 phenotype.19-23 In fact, in SS loss of STAT4 expression appears to be a robust and reliable diagnostic marker for this cancer.21 Upregulation of STAT3 signaling has been consistently documented in advanced stages of CTCL.24-28 During late stages autocrine IL-21 stimulation was shown to be responsible for constitutive activation of STAT3.29 Furthermore, others have suggested that malignant T cells may acquire completely cytokine-independent constitutive activation of JAK3 signaling, which can drive a constitutive activation of STAT3.24,25 Constitutively active STAT3 can increase survival and resistance to apoptosis in malignant T cells, promote sensitization to IL-2 stimulation,24,29 promote Th2 phenotype in advanced disease and induce expression of miR21 oncogenic microRNA.30 STAT3 signaling in CTCL was also shown to upregulate VEGF,31 the anti-inflammatory cytokine IL-1032 and a suppressor of cytokine signaling-3 (SOCS).33 The latter was demonstrated to confer resistance to IFN-a in malignant T cells.32,33 Another important concept that has emerged in recent years is that the phenotype of malignant T cells is not fixed and this cancer exhibits a phenotypic plasticity that is highly sensitive to cytokine milieu in the tumor microenvironment.32 Based on the above described evidence, STAT signaling appears to play a central role in this malignant phenotype switching.
Results Correlation of STAT 4 and 6 expression with Th1, Th2, Treg, Th9, and Th17 phenotypes Previous reports documented that many STAT members regulate Th differentiation phenotypes and determine cytokine expression in CTCL malignant cells.34,35 Hence, we wanted to test the expression of Th1 (IFN-g, IL-2, T-bet and IL-12A) vs. Th2 (IL-4 and IL-5) vs. Th9 (IL-9) vs. Th17 (IL-17A and IL-17F) vs. Treg (TGF-b1 and FOXP3) markers in these cells (Table S1). As demonstrated in Fig. 1A and Fig. S1, most cell lines spontaneously do not express or only weakly express Th1 markers. However, T cell stimulation
2976
with PMA (phorbol 12-myristate 13-acetate) and ionomycin or with CD3/CD28 DynabeadsÒ in select cell lines (e.g. Hut78, HH, H9, SZ4, Sez4 and Hut102) led to an upregulation of a number of Th1 genes (Fig. 1A; Fig. S1). In these cell lines the expression of Th1 markers correlated with the expression of STAT4 mRNA. On the other hand, most cell lines heterogeneously express to some degree IL-4 and IL-5 Th2 markers and in a number of cases such expression corresponded to STAT6 expression (Fig. 1A; Fig. S1). Western blot analysis confirmed the expression of STAT4 and STAT6 genes on the protein level (Fig. 1B). Specifically, cell lines that spontaneously express high levels of STAT6 (e.g., MyLa, PB2B, MaC2A, MJ and Hut102) did not express or only weakly expressed STAT4. On the other hand, cell lines that strongly express STAT4 (e.g., Sez4 and SeAx) did not express STAT6 under non-stimulated conditions. IL-9 (Th9 marker) was only detected in MyLa cells upon stimulation with CD3/CD28 DynabeadsÒ , while most CTCL cell lines were IL-9-negative. Consistent with previous reports,32,36,37 MyLa, PB2B, SZ4, Sez4 Hut102 and SeAx cells express IL-17F, IL-17A or both. Furthermore, these cytokines were further upregulated upon T cell stimulation (Fig. 1; Fig. S1). With respect to Treg phenotype, most malignant cells expressed TGF-b1 to a variable degree, but only MJ, Mac2A, SZ4 and SeAx SS-derived cells expressed detectable FoxP3. Moreover, Fig. 1 demonstrates that in immortalized malignant T cells the molecular definitions between different T helper subtypes are not strictly followed. Unlike, normal T cells, malignant T cell demonstrate molecular overlap across a number of Th phenotypes. Effect of Histone Deacetylase (HDAC) inhibitors on STAT expression in MyLa patient-derived malignant cells Recently, epigenetic changes became a significant focus of basic and clinical research in CTCL. A number of previous studies documented methylation/histone acetylation abnormalities in malignant CTCL cells.38,39 In fact, 2 of the commonly used medications for advanced stages of this cancer are HDAC inhibitors (Romidepsin and Vorinostat).40 Hence, we wanted to test whether histone acetylation mediates the expression of STAT4 and STAT6 thereby potentially affecting the balance between Th1 vs. Th2 phenotype in CTCL. To achieve that we treated MyLa cells for 24 hours with HDAC inhibitors, Romidepsin and Suberoylanilide Hydroxamic Acid (SAHA also known as Vorinostat). MyLa is a commonly used cell line derived from an MF patient skin biopsy.41 As summarized in Table S2, out of 11 cell lines, this line is the only one derived from a skin biopsy of a typical MF patient. At baseline MyLa cells have very weak STAT4 expression and moderate STAT6 expression, which correlates with the advanced disease phenotype (Fig. 1). As demonstrated by RT-PCR, treatment with Vorinostat and Romidepsin upregulated the expression of STAT4 in these cells (Fig. 2A), while concomitantly
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Downloaded by [Brandeis University] at 02:55 25 November 2014
Figure 1. (A) Correlation of STAT4 and STAT6 expression with different T helper phenotype markers in CTCL cell lines under normal control vs. T cell stimulation culturing conditions (e.g., 10 ng/mL of PMA (phorbol 12-myristate 13-acetate) and 1 (M of ionomycin or with CD3/CD28 DynabeadsÒ ). (B) Western blot analysis of STAT4 and STAT6 expression in patient-derived CTCL cell lines under normal control vs. T cell stimulation culturing conditions (i.e., PMA and ionomycin)
downregulating the expression of STAT6 (Fig. 2B). Hence, this finding suggests that there is an inverse relationship in the expression of these genes, which is in part regulated by histone acetylation.
siRNA-mediated knock down of miR-155 leads to upregulation of STAT4 in MyLa cells Oncogenic miR-155 microRNA was recently documented to play an important role in CTCL pathogenesis and was found to be upregulated in advanced stages of this cancer.15,42 Using Bielefeld University Bioinformatic Server database we performed a screening analysis for matches in the STAT4 3’UTR, which identified a putative miR-155 binding site (position 2560 to 2690 of STAT4 mRNA; NM_003151.3). Hence, we wanted to test whether siRNA– mediated knock down of miR-155 expression will lead to the upregulation of STAT4 mRNA. As documented in Fig. 2C, downregulation of miR-155 results in restoration of STAT4 expression in MyLa CTCL malignant cells. This suggests that the observed disease-associated upregulation of miR-155 may be responsible for downregulation of STAT4 and subsequent loss of Th1 phenotype in malignant T cells.
www.landesbioscience.com
Comparison of STAT4 expression between CTCL lesional skin, normal skin from healthy volunteers and skin from patients with benign inflammatory dermatoses Previous reports suggested that expression of certain STAT genes may have diagnostic and prognostic value in CTCL patients.15,16,21 Hence, we wanted to compare the expression of STAT4 by RT-PCR between CTCL lesional skin, normal skin samples from healthy volunteers and biopsy specimens from patients affected by benign inflammatory dermatoses that often mimic CTCL (e.g., psoriasis, chronic eczema and pityriasis rubra pilaris or PRP). As demonstrated in Fig. 3A, STAT4 was preferentially expressed in CTCL lesional skin, but not in normal skin or skin from patients with benign inflammatory dermatoses. It is of interest that upregulation of STAT4 was previously observed in CTCL lesional skin samples,18 when compared to non-malignant inflammatory dermatoses. Further analysis of STAT4 expression in our patient samples documented that this gene was more strongly expressed in patients with stable disease and is downregulated in patients with progressive/advanced disease (Fig. 3B). Using X-Tile software43 we established a cut off
Cell Cycle Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kccy20
Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines ae
ae
be
b
a
Ivan V Litvinov , Brendan Cordeiro , Simon Fredholm , Niels Ødum , Hanieh Zargham , c
c
a
d
b
Yuanshen Huang , Youwen Zhou , Kevin Pehr , Thomas S Kupper , Anders Woetmann & Denis Sasseville a
a
Division of Dermatology; McGill University Health Centre; Montréal, QC Canada
b
Department of International Health; Immunology and Microbiology; University of Copenhagen; Copenhagen, Denmark c
Click for updates
Department of Dermatology and Skin Science; University of British Columbia; Vancouver, BC Canada d
Harvard Skin Disease Research Center; Department of Dermatology; Brigham and Women's Hospital; Harvard University; Boston, MA USA e
These authors have contributed equally to this work. Published online: 30 Oct 2014.
To cite this article: Ivan V Litvinov, Brendan Cordeiro, Simon Fredholm, Niels Ødum, Hanieh Zargham, Yuanshen Huang, Youwen Zhou, Kevin Pehr, Thomas S Kupper, Anders Woetmann & Denis Sasseville (2014) Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines, Cell Cycle, 13:18, 2975-2982, DOI: 10.4161/15384101.2014.947759 To link to this article: http://dx.doi.org/10.4161/15384101.2014.947759
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
REPORT Cell Cycle 13:18, 2975--2982; September 15, 2014; © 2014 Taylor and Francis Group, LLC
Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines Ivan V Litvinov1,y, Brendan Cordeiro1,y, Simon Fredholm2,y, Niels Ødum2, Hanieh Zargham1, Yuanshen Huang3, Youwen Zhou3, Kevin Pehr1, Thomas S Kupper4, Anders Woetmann2,*, and Denis Sasseville1,* 1
Division of Dermatology; McGill University Health Centre; Montreal, QC Canada; 2Department of International Health; Immunology and Microbiology; University of Copenhagen; Copenhagen, Denmark; 3Department of Dermatology and Skin Science; University of British Columbia; Vancouver, BC Canada; 4Harvard Skin Disease Research Center; Department of Dermatology; Brigham and Women’s Hospital; Harvard University; Boston, MA USA y
These authors have contributed equally to this work.
Downloaded by [Brandeis University] at 02:55 25 November 2014
Keywords: Cutaneous T-Cell Lymphoma (CTCL), Mycosis Fungoides (MF), mi-R155, Sezary Syndrome (SS), STAT6 and STAT4 Abbreviations: CTCL, cutaneous T-cell lymphoma; MF, mycosis fungoides; SS, sezary syndrome; HDAC, histone deacetylase; STAT, signal transducers and activators of transcription.
Deregulation of STAT signaling has been implicated in the pathogenesis for a variety of cancers, including CTCL. Recent reports indicate that loss of STAT4 expression is an important prognostic marker for CTCL progression and is associated with the acquisition of T helper 2 cell phenotype by malignant cells. However, little is known about the molecular mechanism behind the downregulation of STAT4 in this cancer. In the current work we test the expression of STAT4 and STAT6 via RT-PCR and/or Western Blot in CTCL lesional skin samples and in immortalized patient-derived cell lines. In these malignant cell lines we correlate the expression of STAT4 and STAT6 with the T helper (Th) phenotype markers and test the effect of Histone Deacetylase (HDAC) inhibitors and siRNA-mediated knock down of miR-155 on STAT4 expression. Our findings demonstrate that STAT4 expression correlates with Th1 phenotype, while STAT6 is associated with the Th2 phenotype. Our results further document that STAT4 and STAT6 genes are inversely regulated in CTCL. Treatment with HDAC inhibitors upregulates STAT4 expression, while at the same time decreases STAT6 expression in MyLa cells. Also, siRNA-mediated knock down of miR-155 leads to upregulation in STAT4 expression in MyLa cells. In summary, our results suggest that loss of STAT4 expression and associated switch to Th2 phenotype during Mycosis Fungoides progression may be driven via aberrant histone acetylation and/or upregulation of oncogenic miR-155 microRNA.
Introduction Cutaneous T-Cell Lymphoma (CTCL) is a rare, but potentially devastating malignancy. Mycosis Fungoides (MF) and its leukemic form, Sezary Syndrome (SS), are the most common variants and account for »50% of all cutaneous lymphomas.1 In Caucasians MF/SS primarily affects individuals over 55 years of age, while in African-Americans, Hispanics and Arabic individuals this disease presents at a significantly younger age (i.e. 20s and 30s).2-4 Multiple epidemiologic studies documented that the incidence of CTCL has increased by »3 fold in the last 25– 30 years.4,5 In the early disease stages, which can last for several years, MF presents as flat erythematous skin patches resembling benign inflammatory diseases, whereas in the later stages, MF cells gradually form plaques or tumors and may disseminate to the lymph
nodes and internal organs.1 The early stages of CTCL are often difficult to distinguish clinically and even histologically from other benign entities including chronic eczema, psoriasis and pityriasis rubra pilaris.6 In advanced disease, malignant cells may appear in the peripheral blood, leading to the leukemic stage of CTCL. A subset of leukemic CTCL, known as SS, is characterized by a triad of erythroderma, lymphadenopathy and detection of malignant T cells with convoluted/cerebriform nuclei on a peripheral blood smear.1 The life expectancy of SS patients is usually less than 3 years.1 The molecular pathogenesis of CTCL remains only partially understood. Recent reports elucidated the nature of cancer initiating cells for MF and SS.7 Multiple studies attempted to clarify the genetic multistep carcinogenesis of CTCL.8-11 Persistent activation of transcription factors of the signal transducers and activators of transcription (STAT) protein family has been
*Correspondence to: Denis Sasseville; Email: [email protected]; Anders Woetmann; Email: mailto:[email protected] Submitted: 05/16/2014; Revised: 07/01/2014; Accepted: 07/03/2014 http://dx.doi.org/10.4161/15384101.2014.947759
www.landesbioscience.com
Cell Cycle
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Downloaded by [Brandeis University] at 02:55 25 November 2014
implicated in the pathogenesis of a wide variety of human cancers, including CTCL. Expression and function of STAT3, STAT4 and STAT5 have been extensively studied in CTCL and these genes appear to play an important role in the disease pathogenesis and can be used as important prognostic markers. Upregulation of STAT5 signaling occurs in the early stages of CTCL.12,13 A growing body of experimental evidence suggests that this gene is important for expression of anti-apoptotic proteins (bcl-2 and bcl-x), cell cycle genes (Cyclin D and c-myc) and oncogenic miR-155 microRNA,14-16 which work together to promote cancerogenesis. miR-155 was dubbed as the “bridge between inflammation and cancer”.17 In the early stages of CTCL, STAT4 appears to be overexpressed, when compared to non-malignant skin samples.18 The expression of STAT4, which is required for T helper (Th) 1 differentiation, is subsequently lost at the later stages, where the disease acquires predominantly Th2 phenotype.19-23 In fact, in SS loss of STAT4 expression appears to be a robust and reliable diagnostic marker for this cancer.21 Upregulation of STAT3 signaling has been consistently documented in advanced stages of CTCL.24-28 During late stages autocrine IL-21 stimulation was shown to be responsible for constitutive activation of STAT3.29 Furthermore, others have suggested that malignant T cells may acquire completely cytokine-independent constitutive activation of JAK3 signaling, which can drive a constitutive activation of STAT3.24,25 Constitutively active STAT3 can increase survival and resistance to apoptosis in malignant T cells, promote sensitization to IL-2 stimulation,24,29 promote Th2 phenotype in advanced disease and induce expression of miR21 oncogenic microRNA.30 STAT3 signaling in CTCL was also shown to upregulate VEGF,31 the anti-inflammatory cytokine IL-1032 and a suppressor of cytokine signaling-3 (SOCS).33 The latter was demonstrated to confer resistance to IFN-a in malignant T cells.32,33 Another important concept that has emerged in recent years is that the phenotype of malignant T cells is not fixed and this cancer exhibits a phenotypic plasticity that is highly sensitive to cytokine milieu in the tumor microenvironment.32 Based on the above described evidence, STAT signaling appears to play a central role in this malignant phenotype switching.
Results Correlation of STAT 4 and 6 expression with Th1, Th2, Treg, Th9, and Th17 phenotypes Previous reports documented that many STAT members regulate Th differentiation phenotypes and determine cytokine expression in CTCL malignant cells.34,35 Hence, we wanted to test the expression of Th1 (IFN-g, IL-2, T-bet and IL-12A) vs. Th2 (IL-4 and IL-5) vs. Th9 (IL-9) vs. Th17 (IL-17A and IL-17F) vs. Treg (TGF-b1 and FOXP3) markers in these cells (Table S1). As demonstrated in Fig. 1A and Fig. S1, most cell lines spontaneously do not express or only weakly express Th1 markers. However, T cell stimulation
2976
with PMA (phorbol 12-myristate 13-acetate) and ionomycin or with CD3/CD28 DynabeadsÒ in select cell lines (e.g. Hut78, HH, H9, SZ4, Sez4 and Hut102) led to an upregulation of a number of Th1 genes (Fig. 1A; Fig. S1). In these cell lines the expression of Th1 markers correlated with the expression of STAT4 mRNA. On the other hand, most cell lines heterogeneously express to some degree IL-4 and IL-5 Th2 markers and in a number of cases such expression corresponded to STAT6 expression (Fig. 1A; Fig. S1). Western blot analysis confirmed the expression of STAT4 and STAT6 genes on the protein level (Fig. 1B). Specifically, cell lines that spontaneously express high levels of STAT6 (e.g., MyLa, PB2B, MaC2A, MJ and Hut102) did not express or only weakly expressed STAT4. On the other hand, cell lines that strongly express STAT4 (e.g., Sez4 and SeAx) did not express STAT6 under non-stimulated conditions. IL-9 (Th9 marker) was only detected in MyLa cells upon stimulation with CD3/CD28 DynabeadsÒ , while most CTCL cell lines were IL-9-negative. Consistent with previous reports,32,36,37 MyLa, PB2B, SZ4, Sez4 Hut102 and SeAx cells express IL-17F, IL-17A or both. Furthermore, these cytokines were further upregulated upon T cell stimulation (Fig. 1; Fig. S1). With respect to Treg phenotype, most malignant cells expressed TGF-b1 to a variable degree, but only MJ, Mac2A, SZ4 and SeAx SS-derived cells expressed detectable FoxP3. Moreover, Fig. 1 demonstrates that in immortalized malignant T cells the molecular definitions between different T helper subtypes are not strictly followed. Unlike, normal T cells, malignant T cell demonstrate molecular overlap across a number of Th phenotypes. Effect of Histone Deacetylase (HDAC) inhibitors on STAT expression in MyLa patient-derived malignant cells Recently, epigenetic changes became a significant focus of basic and clinical research in CTCL. A number of previous studies documented methylation/histone acetylation abnormalities in malignant CTCL cells.38,39 In fact, 2 of the commonly used medications for advanced stages of this cancer are HDAC inhibitors (Romidepsin and Vorinostat).40 Hence, we wanted to test whether histone acetylation mediates the expression of STAT4 and STAT6 thereby potentially affecting the balance between Th1 vs. Th2 phenotype in CTCL. To achieve that we treated MyLa cells for 24 hours with HDAC inhibitors, Romidepsin and Suberoylanilide Hydroxamic Acid (SAHA also known as Vorinostat). MyLa is a commonly used cell line derived from an MF patient skin biopsy.41 As summarized in Table S2, out of 11 cell lines, this line is the only one derived from a skin biopsy of a typical MF patient. At baseline MyLa cells have very weak STAT4 expression and moderate STAT6 expression, which correlates with the advanced disease phenotype (Fig. 1). As demonstrated by RT-PCR, treatment with Vorinostat and Romidepsin upregulated the expression of STAT4 in these cells (Fig. 2A), while concomitantly
Cell Cycle
Volume 13 Issue 18
Downloaded by [Brandeis University] at 02:55 25 November 2014
Figure 1. (A) Correlation of STAT4 and STAT6 expression with different T helper phenotype markers in CTCL cell lines under normal control vs. T cell stimulation culturing conditions (e.g., 10 ng/mL of PMA (phorbol 12-myristate 13-acetate) and 1 (M of ionomycin or with CD3/CD28 DynabeadsÒ ). (B) Western blot analysis of STAT4 and STAT6 expression in patient-derived CTCL cell lines under normal control vs. T cell stimulation culturing conditions (i.e., PMA and ionomycin)
downregulating the expression of STAT6 (Fig. 2B). Hence, this finding suggests that there is an inverse relationship in the expression of these genes, which is in part regulated by histone acetylation.
siRNA-mediated knock down of miR-155 leads to upregulation of STAT4 in MyLa cells Oncogenic miR-155 microRNA was recently documented to play an important role in CTCL pathogenesis and was found to be upregulated in advanced stages of this cancer.15,42 Using Bielefeld University Bioinformatic Server database we performed a screening analysis for matches in the STAT4 3’UTR, which identified a putative miR-155 binding site (position 2560 to 2690 of STAT4 mRNA; NM_003151.3). Hence, we wanted to test whether siRNA– mediated knock down of miR-155 expression will lead to the upregulation of STAT4 mRNA. As documented in Fig. 2C, downregulation of miR-155 results in restoration of STAT4 expression in MyLa CTCL malignant cells. This suggests that the observed disease-associated upregulation of miR-155 may be responsible for downregulation of STAT4 and subsequent loss of Th1 phenotype in malignant T cells.
www.landesbioscience.com
Comparison of STAT4 expression between CTCL lesional skin, normal skin from healthy volunteers and skin from patients with benign inflammatory dermatoses Previous reports suggested that expression of certain STAT genes may have diagnostic and prognostic value in CTCL patients.15,16,21 Hence, we wanted to compare the expression of STAT4 by RT-PCR between CTCL lesional skin, normal skin samples from healthy volunteers and biopsy specimens from patients affected by benign inflammatory dermatoses that often mimic CTCL (e.g., psoriasis, chronic eczema and pityriasis rubra pilaris or PRP). As demonstrated in Fig. 3A, STAT4 was preferentially expressed in CTCL lesional skin, but not in normal skin or skin from patients with benign inflammatory dermatoses. It is of interest that upregulation of STAT4 was previously observed in CTCL lesional skin samples,18 when compared to non-malignant inflammatory dermatoses. Further analysis of STAT4 expression in our patient samples documented that this gene was more strongly expressed in patients with stable disease and is downregulated in patients with progressive/advanced disease (Fig. 3B). Using X-Tile software43 we established a cut off
