Role of Mediator Complex in Breast Cancers

      Role of Mediator Complex in Breast Cancers

Introduction:

Breast Cancers (BCs) are the most common malignant tumors in women with more than 1.8 million new cases registered every year and one of the leading causes of cancer-related deaths in women^1,2. Breast cancers are commonly seen in women but rare and lethal in men³. Distal invasion, metastasis, and drug resistance are causing poor prognosis and lower survival rate in breast cancers¹. Hormone receptor status on the breast cancer cells is used in treatment and to differentiate subtypes of breast cancers. Breast cancers based on the hormone receptor they exhibit could be estrogen receptor-positive breast cancers and estrogen receptor-negative breast cancers, progesterone receptor-positive breast cancers and progesterone receptor-negative breast cancers seen in both benign and malignant tumors^4,5. Estrogen receptors (ER) are activated by the estrogen levels and are essential for the growth of mammary gland development in puberty and during pregnancy⁶. Estrogen receptor-negative breast cancers are seen in patients at younger age than estrogen receptor positive breast cancers and exhibit higher mortality comparatively. However, estrogen receptor- positive breast cancers are more common and comprise about 70% of the total breast cancers⁵. Genetic alterations are one of the main causes of malignant and benign tumors development and survival of the tumors. Anti-estrogen receptor endocrinotherpy is commonly used in the treatment of refractory and metastatic estrogen receptor breast cancers. Adriamycin combination chemotherapy is widely used in treatment of both ER-positive and negative breast cancers. However, long-term use of either endocrinotherapy drugs (up to 50% of patients develop resistance) and/or Adriamycin based drugs exhibit high resistance in BC patients, limiting therapeutic effectiveness and throwing clinical challenges in treatment of breast cancer patients and further causing relapse⁷. Breast cancer treatments evolve rapidly with the advent of new targeted and biological therapies.

Mediator complex subunits has recently emerged as a new target for understanding cancer growth and treatment of those cancers including breast cancers. Alterations and mutations in the genes expressed for MED subunits are implicated with breast cancer carcinogenesis. Mediator is a large multi-protein complex consisting of 30 subunits and acts as a bridge between RNA polymerase II and cell-specific transcription factors. Mediator complex consists of head, middle and tail modules, along with a kinase module which reversibly link with the core modules in transcription process which contribute to transcriptional activation and act as a negative transcription regulator⁸. Mediator complex subunits present in the head module alone could initiate basal transcription but require other modules for activation-dependent transcription.  Transcription is an essential process that involves multiple signaling pathways mainly for growth, proliferation, differentiation, and development of the cells⁸. Mediator complex helps in integrating and transmitting various signals from transcription regulators to RNA polymerase II mediated by different subunits in the mediator complex. RNA polymerase II along with transcription factors transcribes both protein-coding genes and noncoding RNAs like microRNAs. MicroRNAs by gene-specific targeting regulate mRNA expression at post-transcriptional level to cause mRNA degradation or translational repression. MicroRNAs along with Mediator complex is thought to be involved in regulation of ER-positive breast cancers. Individual subunits in the Mediator complex regulate varied sets of genes, while essential subunits are required for all protein-coding genes and nonessential subunits help in regulation and initiation of transcription process⁹.

Mediator complex changes and alterations or dysfunction of transcription machinery impair cell growth, differentiation, development and induce pathological diseases like cancers. Mediator complex is thought to be involved in cell proliferation, tumorigenesis, metastasis and invasion of several cancers including breast cancers⁸. Various subunits present in the Mediator complex are altered and mutated in breast cancers and known to promote resistance to chemotherapy and survival of breast cancers. Knockdown of individual subunits that are overexpressed in breast cancers provide a tool to counter-attack breast cancers and improve therapeutic efficiency of chemotherapeutic drugs used in the treatment of breast cancers. Identification and understanding Mediator complex subunits role in breast cancers provide a novel diagnostic target and as a prognostic marker for breast cancer treatment and identification in imaging techniques and tumor-specific targeted treatment. This review focuses on different subunits in Mediator complex that are involved in breast cancer development and effects of Mediator complex subunits inhibition in breast cancer cells.

Mediator Complex in Breast Cancers: 

Genetic alterations and mutations are one of the causes for the breast cancers, these alterations could be inherited or due to some factors like drugs, growth factors, hormones, kinases, and environmental factors causing genomic changes. Epigenetic changes may also involve in inactivation of tumor suppressor gene and/or uncontrolled cell proliferation and tumorigenesis⁸. Recent studies have shown that genetic mutations and alterations are also seen in Mediator complex subunits of transcription machinery. However, detail knowledge of Mediator complex subunits role in modification, development, and effects on breast cancers is still unclear.

In this review, one subunit from each sub module of Mediator complex that has implications with breast cancers has been chosen. MED19, MED1, MED23, and MED12 from the head, middle, tail and kinase modules respectively were chosen and their involvement in breast cancers are referred from the literature results. MED24 a subunit in the tail module is also referred as it correlates with MED1, MED23, MED16 and other subunits of the MED. Regulating the expression of MED24 can inhibit expression of the subunits that correlates with it and thus MED24 is discussed⁶. Regulating the expression of one or more subunits of the Mediator inhibit breast cancer growth and can also inhibit growth of other cancers.

Figure 1: The figure below shows that MED genes are expressed to form proteins of the Mediator complex. MED is essential for basal transcription and cell survival. Alterations and mutations in MED genes lead to many cancers including breast cancers (MED12 is highly mutated in breast cancers). Overexpression of MED subunits activates TGF-βR, ERα, HER2 expression and methylation of MED subunits by CARM1 leads to resistance to chemotherapeutic drugs. Inhibition of one or more subunits inhibits the signaling pathways for resistance and improve drug sensitivity.

• Fig1: Adapted from: Yin, J. & Wang, G. Development 141, (2014). Streubel, G. et al,. EMBO J. 34, (2015).

MED1:  

MED1 is the first subunit that has been studied to show the association of Mediator and breast cancers⁸. MED1, a subunit in the middle module of Mediator complex is a coactivator of Estrogen receptor alpha (ERα), which plays a prominent role in breast cancers, mediated through estrogen levels. MED1 overexpression is first seen in breast cancers and enhanced Med1 expression increases ERα functions. HER2 (Human epidermal growth factor receptor 2), is overexpressed in breast cancers and known to play a key role in development and resistance of breast cancers. Breast cancer cells that exhibit increased levels of HER2 essentially require MED1 for the expression of E2-ERα (MED1 directly interacts with ERα) and (Epidermal growth factor) EGF-ERα genes that in turn mediate HER2 expression.  Thus, downregulation of MED1 impairs ERα mediated transcription and estrogen-dependent breast cancers growth. HER2 overexpression activates MED1 by phosphorylation and critical sites on MED1 and together HER2 and MED1 involve in developing resistance to tamoxifen an anti-estrogen drug. Knockdown of MED1 down regulates ERα thus HER2 and increase sensitivity to tamoxifen in HER2 overexpressing breast cancers. Altering phosphorylation sites on MED1, HER2 signal blockage can also prevent MED1 mediated ERα expression and could be used as therapeutic targets for breast cancer treatment¹⁰.

MED1/TRAP220 is a common target for ERα and other nuclear receptors, also regulate several miRNAs that are known to be involved in breast cancer development^9,11. Med1 induces oncogenic miRNA cluster miR-191/425 mediated through Estrogen receptor alpha and thus promote cell proliferation, migration, and chemoresistance in breast cancers. Estrogen response elements bind to the ERα and MED1 complex and induce transcription of miR-191, which is also known to promote cell proliferation and migration of breast cancers. Silencing MED1 in breast cancers downregulates miR-191/425 cluster and could be used as a potential target for treating breast cancers and other cancers. MED1 is also overexpressed in other cancers like prostate and lung cancers⁹.

Estrogen receptor-positive breast cancers are commonly seen in post menopause women and obesity is one of the risk factors that promote breast cancers in them. Leptin a neuroendocrine hormone is produced by the obese gene, plays an important role in regulating body weight, appetite and several growth factors in both normal and pathological conditions. Histone acetyltransferase SRC1 and Mediator subunit MED1 are involved in the increase of the proportion of cell cycle in S phase (Synthetic phase) and increase in CYCLIN D1 expression mediated through leptin in breast cancer cells. Inhibition of MED1 and SRC1 reduced CYCLIN D1 expression induced by leptin and thus inhibited cell proliferation in breast cancer cells¹².

MED 12:

The protein expressed by MED12 gene is a subunit in the kinase module of Mediator and mutations in this gene are associated with fibroadenomas and phyllodes tumors^2,13. MED12 gene is highly mutated with more than 700 recorded mutations in cancer mutation database¹⁴. Exome sequencing was used to identify recurrent mutations in breast fibroadenomas and 60% to 73% of breast fibroadenomas exhibit recurrent somatic mutations in MED12 located on chromosome Xq13.1¹³. Missense mutations in MED12 affect MED12 interaction with cyclin C-CDK8/CDK19². Phyllode tumors are fibroepithelial tumors of the breast which rarely transform into malignant tumors. MED12 mutations are highly seen in phyllode tumors irrespective of tumor grade, 37 of 46 phyllode tumors exhibited MED12 mutations¹³. Mediator complex regulates ERα which is encoded by ESR1 (estrogen receptor 1) gene and Mediator also interacts with cohesion complex to promote long range chromosomal interactions. Downregulation of MED12 or SMC3 (cohesion subunit, (Structural maintenance of chromosome protein 3)) inhibit ERα levels indirectly by decreasing ESR1 transcription. SMC3 and MED12 both mutually regulate ERα which plays an important role in breast cancer growth. Regulation of MED12 and SMC3 could be used in treatment of Estrogen receptor-dependent breast cancers¹⁵.

Furthermore, MED12 is involved in developing resistance to chemotherapeutic agents mediated through TGF-βR (transforming growth factor beta receptor) signaling pathway. Knockdown of Med12 in breast cancer cells resulted in developing resistance to several drugs that are used in breast cancer treatment mediated through TGF-βR activation. MED12 correlates with coactivator-associated arginine methyltransferase 1(CARM1) that is overexpressed in human breast cancers and associated with poor prognosis. MED12 is a substrate for CARM1 and binds to the TGF-βR2 to prevent glycosylation and TGF-βR2 delivery to the surface of the cell, thus lower levels of MED12 increase TGF-βR2 on cell surface causing extracellular signal-regulated kinase activation and result in resistance to the drugs. CARM1 methylated Med12 at R1862 and R1912 sites causing mutations which lead to drug resistance. Med12 methylation defects caused upregulation of P21 protein which is associated with poor prognosis to chemotherapy in breast cancer patients. MED12 is positively correlated with CARM1 and enhanced levels of MED12 promoted better prognosis in breast cancer patients¹⁴.

MED19:

The protein encoded by the MED19/Rox3 gene is a subunit in the head module of Mediator complex. MED gene is implicated with growth of various cancers and silencing MED19 inhibits growth of those cancers. MED19 is overexpressed in many cancers including prostate cancer, hepatocellular cancer, bladder cancer, breast cancer, Gastric cancer, Non-small cell lung cancers etc^16,17. MED19 is important for the stability of whole Mediator and in the absence of Med19 the Mediator complex becomes unstable, lack interactions with middle module and exhibit reduced affinity to RNA polymerase II. MED19 plays a vital role in proliferation and development of breast cancers. Hence, MED19 could be used as a potential biomarker for molecular targeted therapy.

MED19 is overexpressed in breast cancers and associated with tumor growth and clinical stage indicating MED19 involvement in breast cancers. Breast cancer cells, when transfected with lentivirus mediated MED19 shRNA (small hair pin RNA), inhibits MED19 expression. Med19 silenced breast cancer cells has shown a significant increase in the proportion of G0/G1 phase (Resting phase) of the cell cycle and attenuated the growth of breast cancer cells invitro. These results show MED19 involvement in the cell cycle and proliferation of breast cancers¹⁸.

Adriamycin (ADM) is widely used chemotherapeutic agent and is mostly used in combination chemotherapy for first-line treatment in breast cancer patients. However, patients develop resistance and Mediator subunit MED19 is involved in developing resistance to Adriamycin mediated through HMGB1 (High Mobility Group Box-1) pathway. HMGB1 is a chromatin binding nuclear protein that plays a major role in breast cancer cell proliferation, invasion and metastasis. Furthermore, HMGB1 promote autophagy, radioresistance and chemoresistance in breast cancers and silencing of HMGB1 suppressed NFϰB/p65 (inflammatory signaling pathway) expression. ADM sensitivity is regulated by HMGB1 expression which is dependent on abnormal expression of MED19 in breast cancers. ADM resistant breast cancer tissues exhibited increased levels of MED19 and MED19 induced autophagy compared to ADM sensitive breast cancer cells. Autophagy is a protective mechanism exhibited by cells for carrying homeostatic functions and it is related to developing chemoresistance in various cancers. Autophagy reduces genome damage and inflammation in cancer cells in response to chemotherapy and radiotherapy thus protecting tumor cells. Inhibition MED19 inhibited autophagy and promotes chemosensitivity to Adriamycin in breast cancers by targeting HMGB1-NFϰB/p65 signaling. HMGB1 inhibition has reduced breast cancer cell proliferation, ADM induced autophagy, and improved drug sensitivity to chemotherapeutic agent ADM and response to radiotherapy⁷.

MED 23:

MED23 is a coactivator and binding target of Estrogen receptor and plays a crucial role in genetic expression of Estrogen Receptor-dependent breast cancers. MED23 is overexpressed, promote growth of Estrogen Receptor breast cancers and involved in developing resistance to an endocrinotherapy drug tamoxifen. Furthermore, MED23 is also associated with clinical stage and poor prognosis to endocrinotherapy in breast cancer patients. Antiestrogenic endocrinotherapy drug everolimus has high affinity to estrogen receptor and acts by targeting estrogen, is used to treat refractory and metastatic breast cancers. Long term use of everolimus in breast cancers develop resistance and HER2 which is overexpressed in breast cancers is associated with MED23 in developing resistance. HER2 and MED23 genes are present on the same chromosome and together they regulate and develop resistance to everolimus. Inhibition of MED23 and HER2 inhibits the expression of estrogen receptor target gene and thus inhibit breast cancer growth¹.

HER2 is overexpressed in 30% of breast cancers and associated with poor prognosis and aggressive disease. MED23 and transcriptional activation domain (TAD) of the activator ESX (epithelial restricted transcription factor) form a complex and regulate HER2 expression. ESX is expressed in mammary glands and activates HER2 gene. Inhibition of ESX-MED23 complex inhibits HER2 expression and thus inhibits breast cancers proliferation. MED23 binds with ESX on the transactivation domain interferences in their interactions inhibits HER2 expression and inhibits HER2 expressed breast cancers¹⁹.

MED24:

MED1 is essential for development of mammary gland and lactation, another Mediator subunit 24 is also involved in the mammary glands development mediated through Estrogen Receptor alpha. ERα is activated in response to the plasma estrogen levels and promote growth of mammary glands in adolescence as well as pregnancy. Mediator complex is stable without MED1, but it is inactive for Estrogen Receptor-mediated transcriptional activation. Med1 and Med24 are involved in normal and malignant epithelial growth of mammary glands and MED23/MED24/MED16 complex enhances the Estrogen Receptor alpha mediated coactivation of MED1.MED23, MED24, and MED16 together play an important role in regulating and enhancing the effects of activators in the transcriptional process. MED23/MED24/MED16 complex regulates the dose dependency mediation between MED1 and MED24. However, the complex is unstable if one of the constituents is deficient. Attenuation of MED24 diminishes the effects of MED1 specifically where MED1 dose is critical, resulting in altered phenotypes particularly where MED1 role is involved such as development of mammary glands⁶.

Conclusion and future perspectives:

The literature suggests the involvement of Mediator complex subunits in the breast cancer growth, tumorigenesis, and metastasis. Mediator has been implicated with the growth of several other cancers and diseases. Alterations and mutations in the MED leads to changes in the transcription and result in several pathogenic diseases including cancers. Several MED subunits are associated with breast cancers which include MED1, MED5, MED12, MED13, MED14, MED19, MED23, MED28, MED30, and CDK8⁸. In this review, one subunit from each submodule of MED that are explored for evidences of their association with breast cancers are documented. Several MED subunits are overexpressed in cancers, but the exact mechanism involved in development and metastasis of these cancers is unclear. For instance, MED1/TRAP220 acts as a coactivator for different hormone receptors including nuclear receptor, estrogen receptor-ERα, but it is implicated with development of both hormonal and non-hormonal cancers^4,11,10. MED1 regulates the expression of estrogen as well as epidermal growth factor (EGF)-dependent ERα which further regulates HER2 and miR191/425 cluster that are associated with breast cancer growth. Inhibition of MED1 inhibits both estrogen-dependent and EGF-dependent breast cancers^9,10.

Mediator is also known to be involved in other physiological functions, for example, MED23 interacts with hnRNPL a splicing factor to regulate post-transcriptional activities like alternative splicing which is implicated in growth of cancers⁸. MED1 and MED23 regulate HER2 expression which is implicated in the development of breast cancer. Recent breast cancer treatments rely on molecular targeted therapy and HER2 is the common target for identifying and treating breast cancers. MED12 and MED19 particularly regulate response to chemotherapeutic drugs of cancers mediated through TGF-βR and HMGB1-NFϰB/p65 signaling, also involved in breast cancer progression^14,7. Hence, MED12 and MED19 could be a target as a bio-marker and for treating breast cancers. Modifying or regulating the expression of one or more subunits that are associated with breast cancer progression can be a new therapeutic strategy for treatment and improving prognosis in breast cancer patients.

References:

1. Lin, B., Zhang, L., Li, D. & Sun, H. MED23 in endocrinotherapy for breast cancer. Oncol. Lett. 13, 4679–4684 (2017).

2. Darooei, M. et al. MED12 somatic mutations encompassing exon 2 associated with benign breast fibroadenomas and not breast carcinoma in Indian women. J. Cell. Biochem. (2018).

3. Yalaza, M., İnan, A. & Bozer, M. Male Breast Cancer. J. breast Heal. 12, 1–8 (2016).

4. Burstein, H. J. et al. Adjuvant Endocrine Therapy for Women With Hormone Receptor–Positive Breast Cancer: American Society of Clinical Oncology Clinical Practice Guideline Update on Ovarian Suppression. J. Clin. Oncol. 34, 1689–1701 (2016).

5. Types of Breast Cancer: Triple Negative, ER-Positive, HER2-Positive. Available at: https://www.webmd.com/breast-cancer/guide/breast-cancer-types-er-positive-her2-positive#1. (Accessed: 28th October 2018)

6. Hasegawa, N. et al. Mediator subunits MED1 and MED24 cooperatively contribute to pubertal mammary gland development and growth of breast carcinoma cells. Mol. Cell. Biol. 32, 1483–95 (2012).

7. Liu, B. et al. Med19 is involved in chemoresistance by mediating autophagy through HMGB1 in breast cancer. J. Cell. Biochem. (2018).

8. Schiano, C. et al. Involvement of Mediator complex in malignancy. Biochim. Biophys. Acta – Rev. Cancer 1845, 66–83 (2014).

9. Nagpal, N. et al. Essential role of MED1 in the transcriptional regulation of ER-dependent oncogenic miRNAs in breast cancer. Sci. Rep. 8, 11805 (2018).

10. Cui, J. et al. Crosstalk Between HER2 and MED1 Regulates Tamoxifen Resistance of Human Breast Cancer Cells. (2012).

11. Zhang, X. et al. MED1/TRAP220 Exists Predominantly in a TRAP/ Mediator Subpopulation Enriched in RNA Polymerase II and Is Required for ER-Mediated Transcription. Mol. Cell 19, 89–100 (2005).

12. Saxena, N. K., Vertino, P. M., Anania, F. A. & Sharma, D. leptin-induced growth stimulation of breast cancer cells involves recruitment of histone acetyltransferases and mediator complex to CYCLIN D1 promoter via activation of Stat3. J. Biol. Chem. 282, 13316–25 (2007).

13. Piscuoglio, S. et al. MED12 somatic mutations in fibroadenomas and phyllodes tumours of the breast. Histopathology 67, 719–29 (2015).

14. Wang, L. et al. MED12 methylation by CARM1 sensitizes human breast cancer cells to chemotherapy drugs. Sci. Adv. 1, e1500463–e1500463 (2015).

15. Prenzel, T. et al. Cohesin is required for expression of the estrogen receptor-alpha (ESR1) gene. (2012).

16. Wei, L. et al. Knockdown of Med19 suppresses proliferation and enhances chemo-sensitivity to cisplatin in non-small cell lung cancer cells. Asian Pacific J. Cancer Prev. (2015).

17. Yuan, H. et al. Knockdown of mediator subunit Med19 suppresses bladder cancer cell proliferation and migration by downregulating Wnt/β-catenin signalling pathway. J. Cell. Mol. Med. 21, 3254–3263 (2017).

18. Cui, X. et al. Suppression of MED19 expression by shRNA induces inhibition of cell proliferation and tumorigenesis in human prostate cancer cells. BMB Rep. 44, 547–552 (2011).

19. Asada, S. et al. External control of Her2 expression and cancer cell growth by targeting a Ras-linked coactivator. 99, (2002).