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Tumor Biology

, Volume 37, Issue 4, pp 4409–4420 | Cite as

Role of estrogen receptor alpha in human cervical cancer-associated fibroblasts: a transcriptomic study

  • Mahesh M. Kumar
  • Sravanthi Davuluri
  • Sridhar Poojar
  • Geetashree Mukherjee
  • Akhilesh Kumar Bajpai
  • Uttam Dungarmal Bafna
  • Uma K. Devi
  • Pramod P. R. Kallur
  • Acharya K. Kshitish
  • R. S. JayshreeEmail author
Original Article

Abstract

Cancer-Associated Fibroblasts (CAFs) are crucial in genesis and progression of tumors; however, cervical CAFs (C-CAFs) are not well characterized. Estradiol (E2) has been implicated as a cofactor in human papillomavirus (HPV)-mediated cervical cancer (CxCa), both in animal models and in women using oral contraceptives; however, the exact role of the hormone is unclear. Human C-CAFs have recently been shown to express estrogen receptor alpha (ER-α). We investigated gene expression patterns in ex vivo cultured early and late stage C-CAFs in the context of E2. CAFs were isolated from four patients with early and two patients with late stage CxCa. ER-α expression in CxCa tissues was localized to stromal fibroblast-like cells and confirmed in ex vivo cultured C-CAFs. Two ER antagonists (ICI 182,780 and Methyl Piperidino Pyrazole) were used to unravel ER signaling in CAFs. Microarray technology was used for expression profiling and validated by quantitative reverse transcription PCR. The transcriptomes of C-CAFs across stages indicated their activated state. C-CAFs had gene expression patterns associated with both pro-tumorigenic and pro-inflammatory signaling. Late-stage C-CAFs compared to those of early stage appeared to be more actively metabolizing and cycling but expressed fewer genes related to immune function. We report differential expression profiles between C-CAFs: early vs. late stage and in the presence of ER antagonists. Both ER antagonists seemed to modulate C-CAF function by down regulating genes associated with cell cycle and metabolism, affecting angiogenesis and cancer progression. This study characterized C-CAFs from early and late stage disease, and experiments with ER inhibitors emphasized the probable importance of canonical ER-α signaling. Interfering with paracrine signaling through fibroblast ER-α is worth exploiting as a targeted therapy in CxCa management.

Keywords

Cervical cancer Cancer-Associated Fibroblasts Estrogen Receptor Alpha Estrogen Receptor antagonists ICI 182,780 MPP-Methyl Piperidino Pyrazole 

Notes

Acknowledgments

The authors wish to thank Swathi U. Lekshmi (Senior Research Fellow, Department of Microbiology, KMIO, Bangalore) for her help with immunohistochemistry, Anita Mahadevan (Additional Professor, Department of Neuropathology, National Institute of Mental Health and Neurosciences) for her help in microphotography, Annapoorni Rangarajan (Associate Professor, MRDG, IISc, Bangalore) for her help during the initial stages of CAF isolation, Mahua Sinha (Assistant Professor, Department of Microbiology, KMIO) and Reeta Mani (Associate Professor, Neurovirology, NIMHANS, Bangalore) for editing the manuscript, Neta Erez (Department of Pathology, Sackler School of Medicine, Tel Aviv University, Tel Aviv) for kindly reviewing the manuscript, and Harsha Gowda (Faculty Scientist, Institute of Bioinformatics, Bangalore) for useful discussions and for improving the manuscript. The authors gratefully acknowledge the financial support of RGCB, Thiruvananthapuram, for part funding of the expenditure towards microarray. Financial support from the Indian Council of Medical Research (ICMR), India (No. 5/13/127/2011/NCD-III), and the Council of Scientific and Industrial Research (CSIR), India, for Senior Research Fellowship to MKM (09/999/(0001)/2009-EMR-I) is also acknowledged.

Compliance with ethical standards

Conflicts of interest

None

Financial competing interests

The research and development division of a commercial organization (Shodhaka LS Pvt. Ltd.) was involved in the research work of SD, AKB, and AKK. However, the participation was independent of the commercial interests of the company. There are no financial competing interests in conducting or reporting this work.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the Institutional Ethics Committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Written informed consent was obtained from all individual participants included in the study.

Supplementary material

13277_2015_4257_MOESM1_ESM.docx (20 kb)
Supplementary Table 1 ERα target genes downregulated in MPP treated C-CAFs (DOCX 20 kb)
13277_2015_4257_MOESM2_ESM.docx (16 kb)
Supplementary Table 2 List of ERα regulated genes downregulated in ICI treated early stage C-CAFs (DOCX 15 kb)
13277_2015_4257_MOESM3_ESM.docx (17 kb)
Supplementary Table 3 List of ERα regulated genes downregulated in ICI treated late stage C-CAFs (DOCX 16 kb)
13277_2015_4257_MOESM4_ESM.docx (52 kb)
Supplementary Table 4 Scoring of stromal fibroblast like cells in the normal cervix and SCC cervix from the Human Protein Atlas (www.proteinatlas.org/) expressing various markers (DOCX 51 kb)
13277_2015_4257_MOESM5_ESM.docx (15 kb)
Supplementary File 1 Primer sequences for validation of gene expression by real time PCR (DOCX 14 kb)
13277_2015_4257_MOESM6_ESM.xls (10.1 mb)
Supplementary File 2 Common genes detected in E2-treated early and late stage C-CAFs (XLS 10348 kb)
13277_2015_4257_MOESM7_ESM.xls (3.5 mb)
Supplementary File 3 Differentially expressed genes (p<0.05) in ER antagonist (ICI and MPP) vs. control (E2) treated C-CAFs (XLS 3544 kb)
13277_2015_4257_MOESM8_ESM.xls (3.7 mb)
Supplementary File 4 Enriched Gene Ontology annotations and pathways (p<0.05) for significantly differentially expressed genes (XLS 3830 kb)
13277_2015_4257_MOESM9_ESM.xls (66 kb)
Supplementary File 5 List of ERα target genes (XLS 65 kb)
13277_2015_4257_MOESM10_ESM.jpg (1 mb)
Supplementary Fig. 1 A: A microphotograph showing squamous cell carcinoma with lobules of tumor cells (t) walled in by lymphoid cells in stroma (s) and cancer associated fibroblasts (caf) in interface. B & C: Staining distribution of ERα expression in fibroblast like cells and tumor infiltrating lymphocytes in stroma by immunohistochemistry on SCC of cervix of stages IB2 and IIIA respectively. Tumor cells however were negative. D: Negative control shows lack of expression in fibroblast like cells and lymphoid infiltrates confirming specificity. Inset: Positive control, ERα expressing carcinoma breast. [A: H & E, B & C: Immunoperoxidase ERα, D: negative control, D inset: ERα positive control] (200× magnification). Images are representative of n=30 (JPEG 1042 kb)
13277_2015_4257_MOESM11_ESM.jpg (80 kb)
Supplementary Fig. 2 Photomicrograph of cervical cancer associated fibroblasts (C-CAFs). A: A representative photomicrograph of CAFs isolated by the enzymatic method, 200× magnification. Pieces of tumor tissue were digested enzymatically and cultured. Adherent fibroblasts were isolated by differential trypsinization. A pure population of CAFs was thus obtained. B: A representative photomicrograph of CAFs after explant culture, 100× magnification. Tumor tissue bits were placed in petri dishes and cultured. Adherent fibroblasts were isolated by differential trypsinization. A pure population of fibroblasts was thus obtained after the 3rd passage. Images are representative of n=26 (JPEG 79 kb)
13277_2015_4257_MOESM12_ESM.jpg (19 kb)
Supplementary Fig. 3a ERα expression on CAFs isolated from fresh tumor tissue by explant culture. RNA was extracted from CxCa CAFs and ERα gene was amplified from the cDNA. Polymerase chain reaction products were analyzed on 1.5 % agarose gels. CAFs expressed ERα (197 bp). Representative gel picture of n=20 (JPEG 19 kb)
13277_2015_4257_MOESM13_ESM.tif (463 kb)
Supplementary Fig. 3b ERα expression on MPP treated CAFs: The expression of ERα was evaluated by real time PCR (TIFF 463 kb)
13277_2015_4257_Fig5_ESM.gif (10 kb)

High-resolution image (GIF 10 kb)

13277_2015_4257_MOESM14_ESM.jpg (107 kb)
Supplementary Fig. 4 Relative expression of lineage specific markers in CxCa CAFs. Mean normalized signal intensity (log2 scale) of lineage specific markers of CAFs [19] as against a keratinocyte marker KRT5 [20] across E2-treated early and late C-CAFs microarray experiments (JPEG 107 kb)
13277_2015_4257_MOESM15_ESM.jpg (261 kb)
Supplementary Fig. 5 Expression pattern of a pro-angiogenic and pro-inflammatory genes in C-CAFs. Mean normalized signal intensity (log2 scale) of pro-inflammatory genes and a proangiogenic gene CYR61 [5] across E2-treated early and late C-CAFs microarray experiments (JPEG 260 kb)
13277_2015_4257_MOESM16_ESM.tif (383 kb)
Supplementary Fig. 6a Real time quantitative reverse transcription PCR validation of microarray: Suppl Fig. 6a: Represents genes prominently expressed in both early and late stage CAFs, some of which were relatively upregulated in late compared to early stage disease (log2 scale) (TIFF 383 kb)
13277_2015_4257_Fig6_ESM.gif (6 kb)

High-resolution image (GIF 6 kb)

13277_2015_4257_MOESM17_ESM.jpg (235 kb)
Supplementary Fig. 6b Differentially regulated genes in late C-CAFs compared to early C-CAFs. n=10 cases each (JPEG 234 kb)
13277_2015_4257_MOESM18_ESM.pdf (6.9 mb)
Supplementary Fig. 7 High-resolution images of immunohistochemically stained tissue sections of normal Cervices and SCC Cervix from the Human Protein Atlas (www.proteinatlas.org/) expressing various markers (PDF 7109 kb)

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Mahesh M. Kumar
    • 1
  • Sravanthi Davuluri
    • 2
    • 3
  • Sridhar Poojar
    • 4
  • Geetashree Mukherjee
    • 5
  • Akhilesh Kumar Bajpai
    • 2
    • 3
  • Uttam Dungarmal Bafna
    • 6
  • Uma K. Devi
    • 6
  • Pramod P. R. Kallur
    • 4
  • Acharya K. Kshitish
    • 3
    • 7
  • R. S. Jayshree
    • 1
    Email author
  1. 1.Department of MicrobiologyKidwai Memorial Institute of OncologyBangaloreIndia
  2. 2.Structural Biology Lab, Centre for Medical Research, School of Bio Sciences and TechnologyVellore Institute of Technology (VIT) UniversityVelloreIndia
  3. 3.Shodhaka Life Sciences Private LimitedBangaloreIndia
  4. 4.Department of RadiotherapyKidwai Memorial Institute of OncologyBangaloreIndia
  5. 5.Department of PathologyKidwai Memorial Institute of OncologyBangaloreIndia
  6. 6.Department of GynecologyKidwai Memorial Institute of OncologyBangaloreIndia
  7. 7.Institute of Bioinformatics and Applied BiotechnologyBangaloreIndia

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