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F1000ResF1000ResF1000ResearchF1000Research2046-1402F1000Research London, UK 10.12688/f1000research.4753.2Opinion ArticleArticlesBreast Diseases: Benign & MalignantGynecological CancersMultiple Endocrine Disorders & NeoplasiasEstrogen as Jekyll and Hyde: regulation of cell death v2; ref status: indexed

Zhou Wen a12Zhu Xiaoxia 31 Braman Family Breast Cancer Institute, University of Miami Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, Fl 33136, USA2 Department of Biological Sciences, Columbia University, New York, 10027, USA3 Molecular Oncology Program, Division of Surgical Oncology, Dewitt Daughtry Family of Surgery, University of Miami Miller School of Medicine, Miami, Fl 33136, USAa gmills@example.net and designed the Figure: WZ XZ. Wrote the paper: WZ XZ.


Competing interests: No competing interests were disclosed.

29 9 2014 2014 3 16125 9 2014 Copyright: © 2014 Zhou W and Zhu X2014This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Sustained estrogenic exposure increases the risk and/or the progression of various cancers, including those of the breast, endometrium and ovary. Unexpectedly, physiological level of estrogen together with a novel IKKα inhibitor BAY11-7082 could effectively induce cell apoptosis in ER-positive breast cancer cells, suggesting combining estrogen with IKKα inhibition may be beneficial for breast cancer patients. This opinion article touches upon the dual role estrogen played in inducing cancer cell death and asks whether use of estrogen in combination with IKKα-targeted therapy would be possible reconsider the newly identified crosstalk between ER and NFκB pathway which can be utilized to switch the effects of estrogen on cell death.

US Department of DefenseW81XWH-11-1-0097This work was partially supported by US Department of Defense pre-doctoral grant W81XWH-11-1-0097 (W.Z.).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Revised Amendments from Version 1
The authors would like to thank the referees for the helpful and constructive critiques of the manuscript. The observations made by the referees were very helpful and we have adapted the manuscript according to their suggestions.We have rewritten part of the introduction, including the part Dr Young pointed out, and added more discussion on Perillo's article in the body of the text.
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Introduction
Our research projects currently focus on the understanding of the interplay between different signaling cascades and estrogen receptor alpha (ER) dependent transcription activation in breast cancer. ER is a master transcription factor frequently elevated in breast cancer patients. ER-positive breast cancers usually occur in up to two thirds of newly diagnosed breast cancer patients. Drugs (such as the antiestrogens tamoxifen and raloxifene) have been very effective in treating ER-positive breast cancers and have saved millions of lives. However, this standard treatment does not work for all ER-positive breast cancer patients. Therefore, we enthusiastically review recent research breakthroughs in the context of our ever-developing understanding on ER function. These new mechanistic insights on the crosstalk between ER and NFκB pathway will lay the ground work for improved clinical approaches to the treatment of this type of breast cancer.

For many years, those of us working in the field were used to looking at estrogen as a mitogen through its genomic function mediated by ER-dependent transcription program. Now we realize that estrogen through ER, in addition to regulating gene expression, crosstalks with many non-genomic signaling pathways involved in cell growth, differentiation and apoptosis. These newly identified interplays may give a different flavor to long conceived mitogenic role of estrogen. In this opinion article, we generally reviewed the changing attitude toward the use of estrogen in the clinic, with a focus on newly discovered pro-apoptotic roles of estrogen when combined with IKKα inhibitors. We also discussed the possibility of estrogen and IKKα inhibitor dual-therapy in ER-positive breast cancer treatment.

Historical perspectives: changing attitudes toward the use of estrogen in the clinic
Sir George Thomas Beatson (1896) use oophorectomy to reduce estrogen level in premenopausal women in order to prevent breast cancer occurrence. It was for the first time to reveal the mitogenic role of estrogen at physiological concentration
1. Half a century later, Haddow
et al. (1944) firstly used a high-dose estrogen to induce tumor regression of hormone-dependent breast cancer in postmenopausal women
2. Therefore, the effect of estrogen is dose-dependent, and thus it is important to take into account the hormonal status of women (pre- or postmenopausal). Huggins
et al. (1952) pioneered adrenalectomy to reduce estrogen level for treating mammary cancer
3. Huggins' work is internationally recognized by the most prestigious Nobel Prize (1966) for the contribution in the development of endocrine therapy in hormone-regulated cancer. Elwood Vernon Jensen (1958) characterized the first receptor for estrogen - estrogen receptor alpha. Soon after these discoveries, extensive mechanistic studies have gained large information about estrogen's physiological functions and carcinogenic roles. The Women's Health Initiative research program (1991) initiated a 15-year study enrolled 161,808 generally healthy women aged 50–79 to evaluate the beneficial effects of postmenopausal hormone replacement therapy on heart diseases, bone fractures, and cancers
4. Due to the increased incidence of breast cancer, stroke, and cardiovascular complications in women treated with estrogen alone or with a combination of estrogen and progesterone, the study was terminated prematurely in 2002. Though extensively studied, the definite understanding of the mechanism of estrogen action always challenges our mind.

The challenge: paradoxical role of estrogen in cell death
Estrogen regulates the proliferation and development of tissues expressing estrogen receptors and ER is mainly expressed in breast epithelium, ovary and endometrium. Thus, estrogen is mitogenic for cultured ER positive breast cancer lines. The mitogenic effects of estrogen at the G1-to-S transition are mediated by the key effectors of estrogen action, c-Myc, cyclin D1 and E2F-1
5–
7. c-Myc expression occurs within 15 min of estrogen stimulation, among the earliest responses to estrogen. Estrogen also rapidly induces cyclin D1 expression. In the G1 phase, estrogen drives E2F-1 expression. Nongenomically, Estrogen binding to the ER stimulates rapid activation of Src and signaling pathways MAPK and PI3K/Akt pathways that affect cell survival
8,
9. Based on these understandings of estrogen action, ER protein is assayed in newly diagnosed breast cancers because it is a clinically useful prognostic factor and predicts responsiveness to ER blocking drugs such as Tamoxifen.

Paradoxically, estrogen induces apoptosis under certain circumstances. As aforementioned, high-dose estrogen was used to induce tumor regression of hormone-dependent breast cancer in postmenopausal women before the introduction of tamoxifen
2. This regimen is of clinical interest, given that long-term treatment of breast cancer with the antiestrogen tamoxifen often leads to drug resistance and that sustained tamoxifen exposure may sensitize breast cancer cells to estrogen therapy
10. The field of the mysterious dual effects of estrogen on apoptosis has not much progress until recently.

Research breakthrough
Perillo Group from the Second University of Naples identified a key player, IKKα in the switch of estrogen action in apoptosis
11. They found ER agonist 17β-estradiol (E2) and IKKα kinase specific inhibitor BAY11-7082 (BAY) in combination can induce apoptosis in ERα positive breast cancer cell line, and dual-therapy now receives more and more attention.

In the journal Cell Death & Differentiation, Perillo
et al. recently reported that the inhibition of IKKα by BAY switched the effect of estrogens on breast cancer cells from anti- to pro-apoptotic, which leads the exploration of therapeutic benefits of estrogen into a new era
11. IKKα is the kinase responsible for histone H3 Ser 10 phosphorylation (H3pS10)
12. H3pS10 is known to inhibit H3 Lys 9 dimethylation (H3K9me2) in a space repulsion model
13. Thus, inhibiting H3pS10 by targeting IKKα facilitates estrogen-triggered ER-dependent recruitment of histone methyltransferase Suv39H1. Histone demethylase LSD1 demethylating the Suv39H1 target sites H3K9me2 was increased concomitantly. LSD1-mediated demethylation process is known to produce reactive oxygen species (ROS) and cause ROS-mediated DNA damaging effects
14. The net results after IKKα knowndown is causing more DNA damages to cancer cells through estrogen triggered ER-dependent Suv39H1 and LSD1 binding to ER target gene promoter (
Figure 1).

Figure 1.  Involvement of IKKα in estrogen-triggered ER-dependent activation of the
pS2 promoter.
Upper panel: chromatin landscape and factors present at the
pS2 (as known as
TFF1) promoter in the presence of IKKα. The pS2 promoter is enriched with nucleosomes (blue and white cylinders) that dwell in positions proximal to the transcription start site (+1 position) and at ER binding sites. Only low levels of histone H3 lysine 9 dimethylation (H3K9me2) exist due to the space repulsion of histone methyltransferase from IKKα residing at the neighboring H3S10 site. RNA polymerase holoenzyme (Pol II) (yellow oval) is present at the proximal promoter region near the transcription start site (TSS, shown by black vertical line) of the pS2 gene. Lower panel: chromatin landscape and factors present at pS2 promoter following IKKα knockdown or IKKα inhibitor BAY11-7082 treatment. Once levels of IKKα have decreased, ER recruits the histone methyltransferase Suv39H1 or demethylase LSD1 proteins to bind within the
pS2 promoter. Once the LSD1 is activated and demethylates its target H3K9me2, it generates reactive oxygen species (ROS) to cause DNA damage effects including base oxidation and nicks results from DNA damage itself and related DNA repair. In sum, the inhibition of IKKα results in the reversion of estrogen triggered anti-apoptotic effects to pro-apoptotic effects.

In short, Perillo group identifies a novel crosstalk between IKKα and estrogen signaling and shows that inhibition of IKKα-mediated histone phosphorylation switch ER-mediated anti-apoptotic effects to ER-dependent ROS-mediated breast cell death, which implicates potential dual-therapy of ER agonist (E2) together with IKKα inhibitor (BAY) in a variety of hormone-regulated cancers. Questions remain as to whether this study using tissue-culture adapted cell lines such as MCF-7 is relevant to clinic practice. The consideration here is mainly based on the well documented limitations of using established cell lines to predict a clinical response, with several compounds that show dramatic tumor killing in cell models failing to yield clinical benefits. Given that established cell lines do not fully reflect the genomic heterogeneity we now appreciate existing in different breast tumors, it will be important to test the same drug combination in a more clinical-relevant sophisticated tumor-stromal microenvironment. This merits further investigation using co-culture between malignant epithelial cells, fat and vascular and inflammatory cells as this likely to influence response to therapy. Additionally,
in vivo studies employing patient-derived xenograft model would be very informative in promoting these novel ideas toward clinical trials. Recognizing the known complication of translating scientific discovery to clinical benefit, the question of whether inhibition of IKKα is a valuable therapeutic strategy in ER-positive breast cancer will have to await more positive results from other model systems and also the further development of additional IKKα inhibitors with minimal potential toxicity and highly bioavailable.

Future perspectives
In the last few years, there have been significant shifts in the attitudes toward the use of estrogen in clinic. Estrogen exhibits a broad range of functions that regulates cell proliferation and homeostasis in many tissues. Despite beneficial estrogen functions, sustained estrogenic exposure increases the risk and/or the progression of various cancers, including those of the breast, endometrium and ovary
15. The International Agency for Research on Cancer (IARC) has listed estrogen as known human carcinogen
16. Now, the success of the combination of E2 and BAY will certainly become an "accelerator" to the alternative use of estrogen in treating cancers and we expect to see more positive pre-clinical and/or clinical results on BAY or other IKKα inhibitors in ER-positive hormone responsive cancers in the near future.

Acknowledgments
The authors thank laboratory members for helpful discussions. The authors thank Drs. S. L. Young and P. Saunders for their constructive comments during the post-publication peer review process. Readers are encouraged to read the reviewers' comments for relevant literature and discussions on whether BAY is also effective in endocrine-resistant breast cancers.

10.5256/f1000research.5741.r6796Referee response for version 2 Young Steven L 1Referee1 Department of Obstetrics & Gynecology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Competing interests: No competing interests were disclosed.

24 11 2014 Version recommendationapproveThe authors have addressed all my concerns.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

10.5256/f1000research.5741.r6389Referee response for version 2 Saunders Philippa 1Referee1 Center for Reproductive Health, University of Edinburgh, Edinburgh, UK
Competing interests: No competing interests were disclosed.

3149326518 Version recommendationapproveThe authors have provided responses to my original comments and incorporated some changes into their manuscript which have improved it.


Suggestions for minor modifications:

Section entitled 'Challenges'

The authors state that estrogen regulates the proliferation and development of tissues expressing estrogen receptors and that 'ER' is expressed in steroid--‐ responsive female tissues such as the ovary, endometrium and breast. The authors further state 'Thus, estrogen is mitogenic for cultured ER positive breast cancer cell lines."

The authors need to amend this paragraph to make it clear that there are two estrogen receptor genes both of which are expressed in breast cancers
1
-
3. Studies on knockout mice have highlighted that expression of the ERα isoform has been identified as critically important to cell proliferation in the uterus and breast
4
,
5. Studies in breast cancer cell lines have highlighted the importance of ERα in both up and down-regulation of cell proliferation and this remains a very active area of research
6
,
7. The authors are encouraged to amend the text of this section changing 'ER' to ERα to make better sense of comments made in the section entitled 'Research Breakthrough'.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

10.5256/f1000research.5075.r6196Referee response for version 1 Saunders Philippa 1Referee1 Center for Reproductive Health, University of Edinburgh, Edinburgh, UK
Competing interests: No competing interests were disclosed.

22 9 2014 Version recommendationapprove-with-reservationsNew ideas about the way oestrogens might affect the growth, differentiation and functional development of cancers, including those of the breast, are always welcome. The authors' Opinion Article questions whether giving oestrogens (rather than the more common anti-oestrogens) may merit exploration as an alternative treatment for breast cancers when provided in combination with agents that alter inflammatory signalling pathways. A limitation of the commentary is that it has focused on data from studies using breast cancer cell lines such as the MCF-7 cells cited in the studies by the Perillo group
1
-
3. A limitation of all studies using tissue-culture adapted cell lines such as MCF-7 is that they do not reflect the genomic heterogeneity we now appreciate exists in different breast tumours
4, only a proportion of which are considered classically activated by oestrogens because they are ER alpha positive. In spite of these reservations the authors do raise some interesting points.

Firstly, they highlight the potential for inflammatory mediators such as those that activate the NFκB signalling pathway as targets for therapies in breast cancer treatment. One example provided is the data from the Perillo group, reporting that inhibition of IKKα kinase specific inhibitor BAY11-7082 might be beneficial in switching the effects of oestrogens from anti- to pro-apoptotic. These studies are in agreement with those from a number of other authors claiming that combination therapies are likely to be beneficial in treatment of this malignancy. Notably, there is an interesting paper by Biswas
et al. published in 2004
5 which examines how NFκB activation in breast cancer specimens might have a role in cell proliferation and apoptosis. Notably, in the Biswas paper, they report results suggesting that activated NFκB is predominantly detected in ER
negative rather than ER positive breast tumours. This paper, as well as the recent genomic proflling of breast cancers from 2000 patients
4 highlights the importance of considering the both the immediate microenvironment of the tumour as well as the adjacent non-tumour tissue. Notably, in considering how oestrogen therapy might be utilised, it is important to take into account the hormonal status of the woman (pre or post menopausal) as well as the compelling data that has demonstrated intra-tissue oestrogen biosynthesis in fat and other cells close to the malignant epithelial cells
in situ
6. A paper that has just appeared in the Journal of Clinical Endocrinology and Metabolism by Savolainen-Peltonen and colleagues
7 is particularly relevant. In this paper, the authors compared the metabolic pathways producing active oestradiol in breast subcutaneous adipose tissue of postmenopausal women with and without cancer, and these showed there were differences depending on whether the adipose tissue was proximal or distal to a tumour. If the tumour is already bathed in high concentrations of oestrogen either from the periphery or local biosynthesis then addition of more ligand is unlikely to be effective.

The local inflammatory environment of the tumour is an important factor in tumour progression and it is influenced by activation of NFκB activity leading to production of inflammatory mediators such as TNFα, IL-1β and prostaglandin E2 from adipocyteassociated macrophages and COX-2 inhibitors have been cited as effective in reducing recurrence of cancer
8. Local increases in inflammatory mediators in turn stimulate adipocyte aromatase expression/activity and hence oestradiol production
9 that may be particularly relevant to obese women
9
,
10. Thus, when considering suppression of NFκB activity in combination therapy there may be many context-dependent impacts on the tumour microenvironment to consider.

The role of NFκB activation as a regulator of inflammatory signalling is particularly important to consider in the context of endocrine resistant breast cancer which cannot be addressed using MCF-7 cells. A new paper in Molecular Cellular Endocrinology by Litchfield
et al. 
11 notes that regulation of endocrine-resistant breast cancer cells may also be mediated by other transcription factors such as COUPTFII. So the keys questions to be addressed in considering the value of the future prospective advanced within this Opinion piece are the following:
Are studies in isolated cell lines such as the one described really relevant when it comes to translating new therapies into practice?

Is inhibition of inflammatory mediators such as that using the BAY compound really most effective in cells that are not actually estrogen responsive?This merits further investigation.

Finally, how can we move forward with the field?

 

In our opinion, an excellent way to advance these studies would be to conduct further investigations using methods which better reflect the microenvironment of an intact tumour
in situ, this should include studies on the interplay between malignant epithelial cells, fat and vascular and inflammatory cells as this likely to influence response to therapy and it cannot be under-estimated that the individual responses to oestrogens by these different cell types may vary considerably. Examples of such studies currently include 3 being explored in the context of breast cancer, including organoids, tissue slices or xenografts. These approaches are more likely than studies in MCF-7 cells to inform choice of new combination therapies for testing in clinical trials; oestrogen combined with NFκB inhibition should be tested in these systems
12
-
14.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

10.5256/f1000research.5075.r5459Referee response for version 1 Young Steven L 1Referee1 Department of Obstetrics & Gynecology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Competing interests: No competing interests were disclosed.

11 8 2014 Version recommendationapprove-with-reservationsIn this opinion manuscript, Zhou and Zhu highlight recent research that suggests estrogen can act on opposing signaling pathways to either promote proliferation or cell death in breast cancer cells and, further, that the signaling can be biased toward cell death by inhibition of IKKa action. The manuscript is thought provoking and timely and the argument is logical and generally well-constructed. The title is eye-catching and appropriate. The figure is attractive and highlights the key point of the manuscript. If the areas of concern (below) are addressed, the conclusions will be justified by the argument presented.

I have four areas of concern:
The manuscript is marred by frequent grammatical and English language usage errors as well as occasional typographic errors. While readable, it is sometimes difficult to determine the precise meaning of the authors' statements. 

In the introduction, the authors state that estrogen, in combination with IKKa, can induce breast cancer apoptosis. However, it is IKKa inhibition that is used, suggesting that it is the absence of IKKa effect that is important - this should be clarified as currently the penultimate sentence of the introduction paragraph appears to be contradicted by the sentence following.

The authors propose that IKKa inhibition has great promise for clinical use, but fail to remark on factors that would be important for feasibility of such therapy (see below):

 
If IKKa were inhibited systemically, would apoptosis be largely limited to the cancer cells? What about other estrogen target tissues?

Are there known toxicities of BAY or other IKKa inhibitors that would need to be considered? 

 Are the concentrations of estradiol and IKKa inhibitor necessary to induce cell death achievable in humans?

Are the concentrations of estradiol required for induction of cell death in the normal pre-menopausal range, or are these pharmacological levels: important implications for treatment of women with functioning ovaries and for the thrombogenic side effects of estrogen therapy, which are dose-related.




4. The abstract does not effectively summarize the manuscript.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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