Why are high EZH2-expressing breast cancer cells prone to bone metastasis?

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Breast cancer is the most commonly diagnosed cancer in female individuals worldwide1. About 50-70% of patients with late-stage breast cancers develop bone metastases that cause skeletal-related events, including pain, pathological fractures, spinal cord compression, hypercalcemia, and other complications2. The treatments for bone metastasis are limited and merely palliative; standard antiresorptive agents, chemotherapy, and radiotherapy can delay or lessen skeletal-related events, but they cannot cure bone metastasis3. Exploring the molecular mechanism of bone metastasis comprehensively may guide the development of new therapeutic strategies for patients with bone metastasis.

Enhancer of zeste homolog 2 (EZH2) is considered a breast cancer oncogene. It is a histone methyltransferase that serves as an enzymatic subunit of the polycomb repressive complex 24. EZH2 regulates gene expression through trimethylation of histone H3 at lysine (K) 27 (H3K27me3) or as a transcription co-factor4,5. It is reported that overexpression of EZH2 is correlated with increased metastases of prostate and breast cancers6,7; we also found that EZH2 expression is negatively correlated with bone metastasis-free survival in patients with breast cancers, therefore, we explored the function of EZH2 in breast cancer bone metastasis.

 

Does EZH2 promote bone metastasis? Can we use EZH2 methyltransferase inhibitor to block EZH2-induced bone metastasis?

To explore the function of EZH2 in breast cancer bone metastasis, we knocked down or knocked out EZH2 in the bone-seeking 231-1566 cell subline of the MDA-MB-231 breast cancer cells, and injected the EZH2-depleted cells or control cells respectively into the left ventricles of nude mice to generate bone metastasis. Mice injected with EZH2-depleted cells had significantly longer bone metastasis-free survival, overall survival, and fewer bone metastases than did mice injected with control EZH2-expressing cells. On the other hand, we re-expressed EZH2 in the EZH2-depleted MDA-MB-231 sublines, and injected intracardiac into mice to induce bone metastasis. Compared to vector control cells, EZH2 re-expression dramatically increased the incidence of bone metastasis, confirming EZH2’s bone metastasis-promoting function. Therefore, we treated mice that were injected with EZH2-high expressing cells with potent, and selective EZH2 methyltransferase inhibitor GSK126 or EPZ-6438 for bone metastasis inhibition. Surprisingly, neither inhibitor can deter bone metastasis incidence and progression. Furthermore, the methyltransferase-defective H689A-EZH2 mutant induced the outgrowth of bone metastasis like that of wild type EZH2. These data demonstrated that EZH2 promotes breast cancer bone metastasis via methyltransferase-independent function, which cannot by blocked by EZH2 methyltransferase inhibitor.

 

How does EZH2 promote bone metastasis?

Breast cancer bone metastasis frequently induces osteolytic lesions, which lead to massive bone resorption and bone fractures8. Osteolytic bone resorption causes secretion of several growth factors, including transforming growth factor beta (TGFβ). TGFβ regulates “the vicious cycle of bone metastasis”9. which designates the feed-forward cycle among bone metastasis cancer cells, osteoblasts, and osteoclasts in promoting both uncontrolled tumor growth and osteoclast activity8,10,11. To test whether EZH2 modulates the vicious cycle of breast cancer bone metastasis, we triple co-cultured breast cancer cells with RAW264.7 preosteoclasts and MC3T3 osteoblasts under TGFβ  treatment which mimic the vicious cycle of bone metastasis microenvironment. The EZH2-knockout cells showed significantly reduced cell growth compared to control cells; the RAW264.7 preosteoclasts co-cultured with EZH2-knockout cancer cells significantly reduced differentiation into mature osteoclasts than those co-cultured with control EZH2-expressing cancer cells. Remarkably, knockout of EZH2 reduced parathyroid hormone-like hormone (PTHLH) mRNA expression, which is the downstream effector of TGF-β, and an essential mediator of the vicious cycle of breast cancer bone metastasis.

 

How does EZH2 evolve TGFβ signaling?

To explore how EZH2 facilitates PTHLH, and TGF-β signaling, we comprehensively deciphered the mechanism by multiple approaches.  We revealed that EZH2 works as a co-factor of RNA Pol II to upregulate ITGB1 transcription, leading to increased integrin β1 protein expression. Integrin b1 activates focal adhesion kinase (FAK), which phosphorylates TGFβ receptor type I (TGFβRI) at tyrosine 182, that enhances the binding of TGFβRI to TGFβ receptor type II (TGFβRII), therefore activates Smad2 and increases PTHLH expression. Remarkably, we first identified the TGFβRI phosphorylation site at tyrosine 182 (pY182) by mass spectrum experiment; then our structural analysis revealed that Y182 of TGFβRI is highly exposed for potential phosphorylation. Upon phosphorylation by FAK, the distance of the negatively charged phosphate group of TGFβRI to the positively charged K381 of TGFβRII becomes much closer, therefore, significantly enhancing binding of TGFβRI to TGFβRII through increased charge-charge interactions.

 

 How to treat EZH2-induced breast cancer bone metastasis?

Since FAK is a key mediator of EZH2-promoted bone metastasis, we treated EZH2-mediated bone metastasis in mice with FAK inhibitor (FAKi) VS-6063, which is currently tested in clinical trials for treating patients with advanced lymphoma or solid tumors. Excitingly, treatment with the FAKi VS-6063 significantly impeded the outgrowth of bone tumors compared to the vehicle control group and did not induce significant side effects. However, EZH2 methyltransferase inhibitor GSK126 treatment did not block tumor outgrowth in the bones.

 

In summary, our data signify integrin β1-FAK as a new downstream effector of EZH2 in breast cancer cells, and EZH2-integrin β1-FAK axis cooperates with TGFβ signaling pathway to promote bone metastasis of breast cancer (Figure 1). Our newly identified tyrosine phosphorylation site at Y182 of TGFβRI is important for regulating the binding of TGFβRI to TGFβRII and subsequent TGFβ/Smad2 pathway activation. EZH2 clearly promotes cancer cell outgrowth in bone metastasis, partially because of the TGFβ-enriched bone microenvironment. In the bone microenvironment, activated TGFβ, along with the enhanced binding of TGFβRI and TGFβRII from FAK-induced TGFβRI-Y182-phosphorylation, builds the critical and distinct bone metastasis vicious cycle. In addition, our findings and previous publications indicate that EZH2 plays distinct functions in different types of cancer and in metastases of different organs, targeting downstream effectors of EZH2, or EZH2’s enzyme function should be carefully evaluated based on the cancer types and metastatic organ sites.  We found that FAK is a downstream effector of EZH2 in the vicious cycle of breast cancer bone metastasis. Thus, treatment with a FAK inhibitor combined with standard antiresorptive agents, chemotherapy, or radiotherapy may provide added benefit to breast cancer patients who suffer from bone metastasis.  

 

High EZH2-expressing cells have strong ability to invade and metastasize from the primary tumor to the bone. In bone-metastatic tumor cells, EZH2 functions as a transcription co-factor to increase ITGB1 transcription. Integrin 1 activates FAK and induce phosphorylation at Y397 of FAK, which phosphorylates TGFRI at Y182. pY182-TGFRI increases binding with TGFRII, thereby activating pS465/467-Smad2, PTHLH expression, and the vicious cycle of breast cancer bone metastasis.

Figure 1. A model of EZH2’s interaction with TGFβ signaling in enhancing breast cancer bone metastasis. High EZH2-expressing cells have strong ability to invade and metastasize from the primary tumor to the bone. In bone-metastatic tumor cells, EZH2 functions as a transcription co-factor to increase ITGB1 transcription. Integrin β1 activates FAK and induce phosphorylation at Y397 of FAK, which phosphorylates TGFβRI at Y182. pY182-TGFβRI increases binding with TGFβRII, thereby activating pS465/467-Smad2, PTHLH expression, and the vicious cycle of breast cancer bone metastasis.

 

 Contributors: Lin Zhang, Jingkun Qu, Dihua Yu. 

 

References:

 

1            Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2019. CA Cancer J Clin 69, 7-34, doi:10.3322/caac.21551 (2019).

2            Fornetti, J., Welm, A. L. & Stewart, S. A. Understanding the Bone in Cancer Metastasis. J Bone Miner Res 33, 2099-2113, doi:10.1002/jbmr.3618 (2018).

3            Rossi, L., Longhitano, C., Kola, F. & Del Grande, M. State of art and advances on the treatment of bone metastases from breast cancer: a concise review. Chin Clin Oncol, doi:10.21037/cco.2020.01.07 (2020).

4            Kim, K. H. & Roberts, C. W. Targeting EZH2 in cancer. Nat Med 22, 128-134, doi:10.1038/nm.4036 (2016).

5            Zhang, L. et al. Blocking immunosuppressive neutrophils deters pY696-EZH2-driven brain metastases. Sci Transl Med 12, doi:10.1126/scitranslmed.aaz5387 (2020).

6            Ren, G. et al. Polycomb protein EZH2 regulates tumor invasion via the transcriptional repression of the metastasis suppressor RKIP in breast and prostate cancer. Cancer Res 72, 3091-3104, doi:10.1158/0008-5472.CAN-11-3546 (2012).

7            Kowalski, P. J., Rubin, M. A. & Kleer, C. G. E-cadherin expression in primary carcinomas of the breast and its distant metastases. Breast Cancer Res 5, R217-222, doi:10.1186/bcr651 (2003).

8            Mundy, G. R. Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer 2, 584-593, doi:10.1038/nrc867 (2002).

9            Kang, Y. Pro-metastasis function of TGFbeta mediated by the Smad pathway. J Cell Biochem 98, 1380-1390, doi:10.1002/jcb.20928 (2006).

10         Zhang, W., Bado, I., Wang, H., Lo, H. C. & Zhang, X. H. Bone Metastasis: Find Your Niche and Fit in. Trends Cancer 5, 95-110, doi:10.1016/j.trecan.2018.12.004 (2019).

11         Esposito, M., Guise, T. & Kang, Y. The Biology of Bone Metastasis. Cold Spring Harb Perspect Med 8, doi:10.1101/cshperspect.a031252 (2018).

 

Lin Zhang

Instructor, MD Anderson Cancer Center