Origins and Actions of Tumor Necrosis Factor α in Postmenopausal Breast Cancer

Abstract

Tumor necrosis factor α (TNFα) has many roles in both physiological and pathological states. Initially thought to cause necrosis of tumors, research has shown that in many tumor types, including breast cancer, TNFα contributes to growth and proliferation. The presence of TNFα—derived from the tumor and infiltrating immune cells—within a breast tumor microenvironment has been correlated with a more aggressive phenotype, and the postmenopausal ER+ subtype of breast cancers appears to strongly respond to its many pro-growth signaling functions. We discuss how TNFα regulates estrogen biosynthesis within the breast, affecting the activity of the key estrogen-synthesizing enzymes aromatase, estrone sulfatase, and 17β-HSD type 1. Additionally, we describe the anti-adipogenic actions of TNFα that are critical in preventing adjacent estrogen-producing adipose fibroblasts from differentiating, ensuring that the tumor maintains a constant source of estrogen-producing cells. We examine how the increased risk of developing breast cancer in older and obese individuals may be linked to the levels of TNFα in the body. Finally, we evaluate the feasibility of targeting TNFα and its associated pathways as a novel approach to breast cancer therapeutics.

Introduction

E strone, estriol, and estrodiol form the biologically active group of hormones collectively known as estrogens (Chen and others 2008). Enzymatically catalyzed in a series of rate-limiting steps from cholesterol in specific tissues, estrogens may circulate as an endocrine hormone or act locally on target cells (Bulun and others 2005). In humans, estrogen acts as the primary female sex hormone, regulating processes related to the growth, differentiation, and physiology of the reproductive system while also exerting its effects in bone, brain, liver, and the cardiovascular system (Pearce and Jordan 2004). Estrogen action also contributes to the pathological states seen in breast, ovarian (Pujol and others 1998), colon (Campbell-Thompson and others 2001), and prostate cancers (Chen and others 2008). The link between estrogens and breast cancer was first postulated as early as 1934 when an early study demonstrated the ability of prolonged exposure to estrogens to cause legions on the breast (Geschickter and others 1934). Work in this area has progressed rapidly since then, and our understanding of the role that estrogens play in breast cancer is now much more advanced. Accompanying this has been the development of a range of therapeutic drugs to target the action of estrogen in breast cancer.

Local estrogen production is a major contributing factor to the risk of developing breast cancer (Miller 2006), with up to 70% of postmenopausal tumors expressing the estrogen receptor and therefore classed as ER+ tumors. Estrogens are strongly associated with continuing development and progression of disease pathology (Manning and others 1990). Many factors have been identified within the breast tumor microenvironment that are able to stimulate increased local estrogen production. One that has risen to prominence recently is the proinflammatory factor tumor necrosis factor-α (TNFα). This cytokine helps to link the role of the invading immune system in tumor development and to explain the altered breast cancer risk that comes with advanced age and obesity (Reed and Purohit 1997). In this review, we explore the molecular basis for TNFα regulation of estrogen biosynthesis in breast cancer and explore how it contributes to disease risk. We also assess the suitability of TNFα as a therapeutic target for ER+ breast cancer treatments.

TNFα: A Pro-Inflammatory, Pro-Cancer Cytokine

A factor that could induce necrosis of tumors in mice was first isolated from serum (Carswell and others 1975; Haranaka and Satomi 1981). The mouse itself was producing TNF from macrophages and lymphocytes, capable of inducing death in a number of cell types not restricted to cancer cells (Granger and others 1969). This discovery was heralded as a potential new therapy for cancer, whereby the growth of a tumor could be retarded using this molecule. Consequently, the human genomic clone was quickly isolated and the cDNA and protein sequence found to be 80% homologous to the mouse TNFα (Marmenout and others 1985; Pennica and others 1984; Shirai and others 1985). Although systemic toxicity proved to be an insurmountable barrier to the use of TNFα as an anti-tumor agent, the study of this molecule and its related TNF superfamily members has remained important to the understanding of apoptosis, immune defenses, inflammation, and cancer processes (Locksley and others 2001).

In humans, TNFα is produced primarily by the macrophages and monocytes of the immune system (Kornbluth and Edgington 1986), but production has also been detected in lymphoid cells, endothelial cells, neuronal tissue, adipose fibroblasts, and tumor cells (Wajant and others 2003). TNFα signals via 2 receptors: type 1 (TNFR1) and type 2 (TNFR2). Both bind TNFα with equal high affinity (Grell and others 1995; Grell and others 1998); however, differences lie in their tissue distribution. TNFR1 is constitutively expressed in most tissues and is thought to be the primary mediator of TNFα signaling in the body. TNFR2 expression has thus far been isolated to the cells of the immune system, where its role is more pronounced in the lymphoid system (Wajant and others 2003). As a consequence, much more is understood about signal transduction via TNFR1.

TNFα has the unique property among its related family members in that it displays proinflammatory effects in addition to its proliferation and apoptotic abilities (Tracey and others 1986). The wide ranging effects of TNFα on a number of different cell types have made it an intently studied target not only for disease therapies but also for understanding basic cellular processes and their regulation.

TNFα and Breast Cancer

As a potent signaling molecule, TNFα has been found within the microenvironment of several cancer types, including pancreatic (Karayiannakis and others 2001), renal (Yoshida and others 2002), prostate (Pfitzenmaier and others 2003), kidney, lung, bladder, oesophageal, melanoma, and leukemia (Mantovani and others 2000). In a breast tumor environment, increased concentrations of TNFα have been detected in the breast cyst fluid and breast tumor cytosol, both of which are known to stimulate estrogen production (Macdiarmid and others 1994). Furthermore, the presence of TNFα has been strongly correlated to a metastatic, invasive breast tumor phenotype (Miles and others 1994; Leek and others 1998). Serum concentrations of TNFα are also higher in patients with more advanced breast cancers (Sheen-Chen and others 1997), and can be used as a predictive marker for response to chemotherapy treatments (Berberoglu and others 2004). This observation is consistent with clinical findings and animal studies suggesting that endogenously secreted TNFα in the presence of a tumor exerts a proliferative response rather than tumor regression (Mochizuki and others 2004; Zarovni and others 2004), contrary to the initial isolation of TNFα as a potential anti-cancer agent.

TNFα appears to be particularly critical to the development and progression of the estrogen-responsive breast tumor type. The activity of key estrogen-biosynthesis enzymes have been shown to be increased by TNFα in adipose tissue, namely, aromatase (Purohit and others 2002), estrone sulfatase (Newman and others 2000), and 17β-hydroxysteroid dehydrogenase type 1 (Duncan and others 1994). TNFα can also modulate expression of genes in ER+ breast cancer cell lines that lead to the promotion of cell growth and proliferation, metastasis, loss of cell cycle control, and degradation of the extracellular matrix (Jeoung and others 1995; Yin and others 2009; Li and others 2012).

Sources of TNFα Within the Breast Tumor Microenvironment

Heightened concentrations of TNFα are found within the breast tumor microenvironment, with several different cell types thought to contribute to cytokine production. Tumor-associated macrophages and lymphocytes, which constitute up to 50% of the total tumor volume (Kelly and others 1988), are thought to be the primary contributors. TNFα is associated with immune response, and in the healthy body is mainly produced by cells of the immune system (Kornbluth and Edgington 1986). Tumor epithelial cells secrete chemo-attractants such as Interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), which leads to an invasion of immune cells into the breast tumor matrix (Leonard and Yoshimura 1990; Baggiolini and others 1994). These immune cells in turn respond to soluble factors secreted from breast cancer cells by upregulating their production of TNFα (Eichbaum and others 2011). Conditioned media from cultured monocytes and lymphocytes of breast cancer patients are able to stimulate aromatase activity in human breast adipose fibroblasts, and this can be attributed to the high levels of TNFα also detected in the media (Singh and others 1997).

Evidence has suggested that tumor cells are themselves able to produce TNFα in order to achieve proliferative advantage, although this has not been confirmed of breast cancer epithelial cells. However, several of the main factors known to stimulate TNFα production are present within a breast tumor microenvironment, suggesting that TNFα secretion by tumor epithelial cells is a contributing factor to disease pathology. TNFα is able to positively auto-regulate its own expression in murine 3T3-L1 preadipocytes (Neels and others 2006), and given the abundance of the cytokine surrounding tumor epithelial cells it is possible that it upregulates its own expression. Estrogen increases the expression and secretion of TNFα in cells in uterine and lactotrope cells (De and others 1992; Zaldivar and others 2011), and while this has not been confirmed in breast cancer cells, raises the possibility that a positive feedback loop between TNFα and estrogen exists within a breast tumor microenvironment. Supporting this hypothesis is the finding that estrogen increases the expression of the TNFα receptor TNFR1 in human breast adipose fibroblasts, thereby potentiating the response of these cells to TNFα (Deb and others 2004).

Epigenetic regulation, specifically DNA methylation, is another poorly defined area of TNFα in the context of ER+ breast cancer. It has been established in neuronal cells, hematopoietic stem cells, and macrophages that TNFα expression is under epigenetic regulation (Sullivan and others 2007; Pieper and others 2008). Indeed, a screen for genes differentially expressed following DNA demethylation with 5-aza-2′-deoxycytidine showed that TNFα and many of its target genes showed increased expression (Kim and others 2012). Epigenetic mechanisms are known to factor in the regulation of estrogen biosynthesis as well as many other key breast cancer processes (Knower and others 2010), and the elucidation of its role in TNFα regulation is critical to our overall understanding of the importance of DNA methylation and histone modifications.

TNFα Regulation of Estrogen Biosynthesis

Estrogen biosynthesis

The process of biological estrogen production begins in the mitochondrion, where cholesterol is enzymatically converted to active estrogens through a number of rate limiting steps. The activity of 3 main enzymes is responsible for the production of biologically active estrogens: estrone sulfatase, estradiol-17β-hydroxysteroid dehydrogenase type 1 (17β-HSD type 1), and aromatase. Aromatase converts androstenedione to estrone via an aromatisation reaction, while estrone sulfatase forms estrone from the estrone sulfate precursor. 17β-HSD type 1 then catalyzes the reduction of estrone into estrodiol, the biologically active form of estrogen (see Fig. 1).

FIG. 1.

Tumor necrosis factor-α (TNFα) and its effect on the intratumoral estrogen biosynthesis pathway. Schematic representation of the enzymatic conversion from androgens to active estrogens, and the stages at which TNFα is thought to play a key regulatory role.

Aromatase

Aromatase is the key enzyme responsible for the conversion of androgens to estrogens. Its highly tissue-specific expression is regulated at the transcriptional level of its encoding gene CYP19A1 via tissue-specific promoters (Agarwal and others 1995). In premenopausal women, aromatase expression primarily localizes to the ovary. Here, CYP19A1 expression is mediated via the proximal cAMP-responsive promoter II in response to FSH signaling (Means and others 1991; Jenkins and others 1993). Once a woman reaches menopause, the ovaries cease estrogen production, and the source of oestrogen biosynthesis switches primarily to the adipose tissue. This occurs largely in breast adipose, with a low level of aromatase expression maintained in this tissue via the use of distal promoter I.4 (PI.4) (Zhao and others 1995a). PI.4 activity can be stimulated in vitro by cytokines such as TNFα, oncostatin M (OSM), IL-6, or IL-11 (Zhao and others 1995b, 1996) in conjunction with the synthetic glucocorticoid dexamethasone (DEX) (Simpson and others 1981).

OSM, IL-6, and IL-11 have previously been demonstrated to act via the Jak/STAT pathway and the upstream GAS element within PI.4 in breast adipose fibroblasts (Zhao and others 1995b). The mechanisms by which TNFα is able to activate PI.4 are not yet clear, although a previous study has suggested that an upstream AP-1 element may be involved (Zhao and others 1996). While it has been shown many times in vitro, attempts to obtain in vivo evidence of a cytokine-glucocorticoid interaction have not been successful. Studies on women given DEX during therapy found that aromatase activity and, consequently, estrogen conversion were not enhanced (Reed and others 1986). Similar studies in monkeys gave the same result (Longcope 1987). In vitro, TNFα has been shown to induce CYP19A1 PI.4 activity in pachytene spermatocytes (Bourguiba and others 2003) and EM1 endometrial cells (Salama and others 2009) without the presence of DEX; however, again this has not been shown in vivo. The evidence points to a critical role for TNFα in aromatase regulation, and elucidating its mode of action will be important in the treatment of estrogen-driven breast cancers and other diseases.

Estrone sulfatase

Following aromatisation of androstenedione, most of the resulting estrone is converted to estrone sulfate, which is the primary circulating form of estrogens as it has a much longer half-life than other forms (Ruder and others 1972). Estrone sulfate is thought to form a reserve of biologically inactive estrogens ready to be converted into the active hormone firstly through its conversion back to estrone via the action of estrone sulfatase (Purohit and others 2002). Expression of estrone sulfatase mRNA is reported to be robust in most breast tumors when compared with normal breast, and this is associated with a larger tumor volume and poorer prognostic outcomes (Utsumi and others 1999a, b, 2000; Suzuki and others 2003; Honma and others 2006). In addition to increased expression, activity of estrone sulfatase is heightened in breast cancer tissue compared with healthy breast (Santner and others 1993; Evans and others 1994).

Little is known about the role that cytokines, and in particular TNFα, may play in the regulation of estrone sulfatase expression and activity in the breast tumor microenvironment. TNFα, along with IL-6, increases the activity but not transcript levels of estrone sulfatase in primary human fibroblasts derived from both normal and malignant breast tissue (Purohit and others 1996). This suggests that these cytokines are involved in the post-translational modifications of the active enzyme rather than its transcriptional regulation (Purohit and others 2002). Recently, however, it was demonstrated in the ER+ breast cancer cell line MCF7, the ER- breast cancer cell line MDA-MB-231, as well as 2 prostate cancer cell lines that treatment with TNFα significantly increased mRNA expression of estrone sulfatase via the PI-3 kinase/Akt pathway (Suh and others 2011). Therefore, targeting the TNFα-mediated increases in estrone sulfatase transcription and activity could potentially reduce the bioavailability of estrogens to an ER+ breast tumor.

17β-HSD type 1

Estrone must be reduced to estrodiol in order to achieve its full biological activity, and this is mediated through the action of 17β-HSD type 1 (Dumont and others 1992). Expression of 17β-HSD type 1 is strongly correlated with ER+ as well as progesterone receptor-positive breast cancers, indicating local synthesis of estrodiol (Ariga and others 2000). Overall metabolism of estrogens favors inactivation of estrodiol over conversion of estrone to estradiol; however, within tumor tissue there is preferential reduction to the biologically active estrodiol (Beranek and others 1984). This suggests that factors within the tumor are preferentially driving the formation of estrodiol, and TNFα may be a contributing factor. An early investigation into the role of cytokines in breast cancer revealed that TNFα stimulates the conversion of estrone to estrodiol in MCF7 breast cancer cells (Duncan and others 1994). TNFα also increases the activity of 17β-HSD type 1 in the ER+ cell line T47D and ER- cell line MDA-MB-231 (Duncan and Reed 1995), indicating that it acts in a variety of tumor microenvironments. Outside of the breast, TNFα has also been found to stimulate activity of 17β-HSD type 1 in endometrial glandular epithelial cells (Salama and others 2009). Taken together, these finding indicate that TNFα plays a critical role in vivo to increase the bioavailability of active estrogens.

Anti-Adipogenic Actions of TNFα in the ER+ Breast Tumor Microenvironment

As well as promoting estrogen biosynthesis in cancer-associated fibroblasts, TNFα also plays a major role in maintaining these fibroblasts in an undifferentiated state, thus ensuring a constant source of estrogen-producing cells for the tumor.

The desmoplastic reaction is critical in maintaining estrogen supply to the ER+ breast tumor (Deb and others 2004). This dense layer of undifferentiated fibroblasts immediately adjacent to the malignant epithelial cells gives such tumors their characteristic hard consistency (Bianco and others 1995), and many factors secreted by the tumor are directed toward maintaining this layer of undifferentiated cells. This is important in breast tumor pathology as only undifferentiated fibroblasts maintain the capacity to express aromatase and produce active estrogens, a feature lost once differentiation into mature adipocytes occurs (Clyne and others 2002). Accumulation of preadipocytes does not appear to be the natural reaction to growth stimuli, since mouse 3T3-L1 preadipocytes treated with a combination of known growth factors initially proliferate, but eventually differentiate into mature adipocytes under the same conditions (Schmidt and others 1990; Boney and others 1998). ER+ breast tumors must therefore both cause an accumulation of preadipocytes and at the same time prevent them from differentiating into mature adipocytes. TNFα appears to be a major driver of this process (Chae and Kwak 2003).

The two critical transcription factor families that commit the preadipocyte toward differentiation in to a mature fat cell and maintain its phenotype are CCAAT/enhancer binding protein (C/EBP) and peroxisome proliferator-activated receptor-gamma proteins (PPARs) (Cao and others 1991; Brun and others 1996). These proteins work to transactivate adipocyte-specific gene expression, and inhibition of their expression or activity can lead to inhibition of preadipocyte differentiation as stimulated by known differentiation factors, or dedifferentiation of committed adipocytes (Tamori and others 2002). Two key family members are C/EBPα and PPARγ, which together act synergistically to drive mature adipocyte differentiation (Meng and others 2001). TNFα can repress the expression of both C/EBPα and PPARγ in preadipocytes, directly inhibiting their capacity to differentiate (Zhang and others 1996). This has been demonstrated in 3T3-L1 cells, mouse adipose tissue, and primary human mammary adipocytes (Hu and others 1995; Chae and Kwak 2003; Guerrero and others 2009). The source of this TNFα appears to be from the tumor epithelial cells themselves, as conditioned media collected from MCF7 and T47D breast cancer cell lines show an enrichment of the cytokine. Treatment of 3T3-L1 preadipocytes with this conditioned media leads to a reduction in C/EBPα and PPARγ expression (Guerrero and others 2009). Melatonin, a naturally secreted hormone from the pineal gland, has been shown to interfere with this process, down regulating the expression of TNFα in epithelial cells while inhibiting aromatase expression in the surrounding fibroblasts (Alvarez-Garcia and others 2012; Knower and others 2012). Further elucidation of other factors capable of restricting TNFα secretion from epithelial cells and therefore its role in the desmoplastic reaction will need to be uncovered in order to improve options for breast cancer therapy.

Clinical Associations Between TNFα and Breast Cancer Risk

Advanced age and TNFα

Advanced age is a major risk factor in the development of breast cancer. Approximately 70% of postmenopausal cases are diagnosed as ER+ tumors, suggesting that processes within the postmenopausal endocrine system are altered as such that peripheral estrogens, particularly those in the breast, are being upregulated. Increased levels of plasma TNFα in older individuals may help to provide an explanation.

As well as being a critical pro-inflammatory immune cytokine, TNFα is also implicated in a number of disease pathologies. These include rheumatoid arthritis (Maini and Taylor 2000), inflammatory bowel disease (Bruin and others 1995), osteoporosis (Fujita and others 1990), and atherosclerosis (Fukuo and others 1997). Most of these conditions affect older individuals, suggesting that increasing concentrations of TNFα in those with advanced age contribute to common diseases associated with aging. Breast cancer could be one other such disease. Animal models were initially investigated to establish a link between aging and increasing TNFα levels. Aged mice and rats show a significantly increased secretion of cytokines from the T-helper cells of their immune systems, and this is likely to account for the increased peripheral estrogen synthesis also observed (Chorinchath and others 1996; Morin and others 1997). A number of studies have since examined this association in a large human cohort and uncovered similar associations between increasing age and higher levels of measured serum TNFα (Paolisso and others 1998).

Obesity and TNFα

In addition to advanced age, rates of breast cancer occurrence are significantly higher in obese women, with increased weight strongly associated with a higher risk of developing not only breast but many other forms of cancer (Basen-Engquist and Chang 2011). Obese breast cancer patients also show higher mortality rates, greater metastasis to distal sites, larger tumor mass, and overall poorer prognosis when compared to non-obese breast cancer patients (Maruthur and others 2009; Hauner and others 2011). Again, strong associations between TNFα and obesity may help to provide an explanation.

In addition to being produced by cells of the immune system, TNFα is also produced in adipose tissue, including mature adipocytes, stromal-vascular cells, and preadipocytes (Hube and others 1999; Weisberg and others 2003; Fain and others 2004). Initially shown in animal models (Hotamisligil and others 1993), TNFα levels are also markedly increased in the adipose tissue of obese individuals (Hotamisligil and others 1995). This has been shown clinically as obese patients record a higher serum concentration of TNFα than age-matched healthy weight individuals. This effect was decreased upon surgery-mediated weight loss (Hotamisligil and others 1995). TNFα may indeed be one of the driving forces behind obesity and insulin resistance, as mice lacking a functional TNFα protein or receptor are protected from diet-induced obesity and insulin resistance (Schreyer and others 1998; Nieto-Vazquez and others 2008). The mechanism resulting in increased TNFα production in states of obesity is, however, undefined, and this knowledge may help explain why obese individuals are at higher risk of breast cancer. It has been recently been shown that TNFα positively regulates its own transcription and secretion in adipose tissues, perhaps explaining how high levels of the cytokine are maintained in obesity (Neels and others 2006).

Significantly, adipose tissue is also the major site of estrogen conversion in postmenopausal women, highlighting a link between increased TNFα and estrogen production. The increased risk of developing breast-cancer in obese women may therefore not only be associated with the increased estrogen production from the higher volume of fat cells, but also with the increased production of TNFα which may further drive estrogen production. Studies in mice have shown that obesity is associated with increased aromatase activity and TNFα expression in the mammary gland (Subbaramaiah and others 2011), supporting this hypothesis.

The Potential for TNFα-Targeted Breast Cancer Therapies

Given its important role in many facets of breast cancer development, progression, and maintenance, TNFα represents an attractive yet challenging therapeutic target. As demonstrated, TNFα plays vital roles in maintaining and upregulating local estrogen biosynthesis as well as preventing the differentiation of estrogen-producing preadipocytes into mature adipocytes. Reducing the capacity of the breast tumor to do either of those things would severely restrict its growth and proliferative potential.

Numerous clinical trials have already investigated the effectiveness of anti-TNFα therapies for the treatment of a number of associated diseases such as septic shock, rheumatoid arthritis, Crohn's disease, and even multiple sclerosis (Shimamoto and others 1988; Elliott and others 1993; van Dullemen and others 1995; Hohlfeld 1996) (see Table 1). Currently used in the clinic are a number of approved monoclonal antibodies as well as a soluble TNFα receptor for the treatment primarily of rheumatoid arthritis patients (Thalayasingam and Isaacs 2011). Concerns were initially raised about their potential to increase a patient's risk of developing certain forms of cancer; however, it is now clear that cancer incidence and prognosis were no worse in patients treated with anti-TNFα therapies compared to those who had not received the treatment (Askling and others 2005; Raaschou and others 2011). Due to its importance in the immune response, the most significant risk appears to be in susceptibility to infectious diseases. Indeed, rheumatoid arthritis patients treated with anti-TNFα therapies appear to be at a greater risk of developing skin infections, soft-tissue infections, and septic arthritis (Dixon and others 2006; Galloway and others 2011). The risk appears to be higher in the first six months of treatment, and is enhanced with advanced age and concurrent use of glucocorticoid treatments (Askling and others 2007; Strangfeld and others 2011). Tuberculosis, listeria, salmonella, and legionella infections also appear at a higher rate in anti-TNFα-treated patients (Dixon and others 2006, 2010).

Table 1.

Current Anti-Tumor Necrosis Factor-α Drug Therapies and Their Applications

	Inflaximab	Adalimumab	Golimumab	Etanercept	Certolizumab
Type¹	Monoclonal antibody	Monoclonal antibody	Monoclonal antibody	P75TNFR/Fc fusion protein	PEGylated humanized Fab fragment
Recognizing ligands¹	TNF	TNF	TNF	TNF and LTα3	TNF
Molecular weight¹	150 kDa	150 kDa	150 kDa	150 kDa	95 kDa
Half-life (days)¹	8–10	10–14	12±3	3	14
Uses	Crohn's disease,² rheumatoid arthritis,³ psoriatic arthritis,⁴ psoriasis⁵	Crohn's disease,⁶ rheumatoid arthritis,⁷ psoriasis⁸	Rheumatoid arthritis,⁹ psoriatic arthritis¹⁰	Rheumatoid arthritis,¹¹ psoriatic arthritis,⁴ plaque psoriasis⁸ and ankylosing spondylitis¹²	Crohn's disease,¹³ rheumatoid arthritis¹⁴

Summary of currently available anti-TNFα therapies. References as indicated within the table in superscript: ¹Thalayasingam and Isaacs (2011), ²van Dullemen and others (1995), ³Harriman and others (1999), ⁴Woolacott and others (2006), ⁵Kirby and others (2001), ⁶Bultman and others (2012), ⁷den Broeder and others (2002), ⁸Chastek and others (2013), ⁹Zhou and others (2007), ¹⁰Yang and others (2011), ¹¹Wakabayashi and others (2012), ¹²Dougados and others (2012), ¹³Schreiber (2011), ¹⁴Weinblatt and others (2012).

TNF, tumor necrosis factor.

TNFα-targeting monoclonal antibodies have so far been demonstrated to retard mouse mammary tumor growth in vivo, as well as inhibiting proliferation of skin cancer cells (Scott and others 2003). Furthermore, TNFα null mice show lower rates of induced tumor formation than the wild type, with TNFα-neutralizing antibodies again able to slow rates of tumor growth (Warren and others 2009). This demonstrates a potential for the use to anti-TNFα therapies in the clinic. TNFα is a ubiquitous cytokine, and its critical role in the pro-inflammatory immune response means that it is present in many tissues as well as circulating plasma. To target its actions in breast cancer would therefore require a therapy to limit undesirable side effects. Indeed, a case of primary breast tumor has been reported in a patient undergoing long-term anti-TNFα treatment for rheumatoid arthritis, suggesting that tumor development not regression may in fact result from anti-TNFα therapies (Pattanaik and others 2011). Further work to elucidate the precise molecular pathways by which TNFα acts and how it is produced to excess within a breast tumor microenvironment is required so that further downstream components may be targeted. For example, agents stabilizing microtubules in breast fibroblasts inhibit TNFα-induced aromatase activity (Purohit and others 1999). Identifying other similar pathways would result in a more specific blockade of TNFα action in breast cancer without compromising its critical immune function.

Summary and Future Perspectives

The research presented in this review highlights the many complexities of the role TNFα has to play within the breast tumor microenvironment. Not only is it implicated in the transcription and activation of key estrogen-producing enzymes aromatase, estrone sulfatase, and 17β-HSD type 1, but it plays a key part in maintaining supporting stroma adjacent to the tumor epithelial cells in an undifferentiated state so that they may continue to produce estrogen. It is found in abundance within the breast tumor microenvironment, with the tumor itself thought to contribute to its production (Fig. 2).

FIG. 2.

Model of TNFα formation and action within the breast tumor microenvironment.

Our understanding of the molecular basis for the actions of TNFα in breast cancer is more developed, but many questions are yet to be answered. For example, it has been well established that TNFα induces expression of the aromatase gene CYP19A1 and that this is via its adipose-specific promoter. What has not been identified is by which signal transduction pathway this is occurring, and what specific transcription factors and cis-acting elements are being activated to initiate this response. Defining the exact mechanisms by which TNFα stimulates aromatase transcription is important if we are to target more specifically in the breast this key estrogenic enzyme. Our understanding of how TNFα contributes to the upregulation of the other estrogen-forming enzymes is not much more advanced, for while their increased activity has been correlated with the presence of TNFα, whether this key cytokine is acting at the transcriptional or translation level and how this is occurring has not been uncovered. Estrogen formation is a precisely regulated process involving many genes and enzymes, and consideration of all components of the pathway must be given in order to effectively limit hormone production.

Targeting TNFα formation and action has also been proposed as a potential novel method for treating ER+ breast cancer (Reed and Purohit 1997). This would not only contribute to lowering the production of estrogen in the stromal cells, but effect the desmoplastic reaction so critical to the tumor's pathology. As discussed, TNFα sequestering monoclonal antibodies are in clinical use for the treatment of several inflammatory-related diseases. Although mouse models have shown a positive response to TNFα antibodies with respect to slowed breast tumor growth, such a treatment has to date not been trailed in humans specifically for the treatment of breast cancer. The effects an anti-TNFα treatment would have on the immune system need to be considered, as increased rates of infection have been shown when TNFα is targeted as a signaling molecule.

In conclusion, TNFα is a complex cytokine implicated in not only the pathology of ER+ breast cancer but also in the risk of developing the disease. It presents an attractive target for therapeutic intervention due to its multi-function role in the tumor microenvironment; however, our limited understanding of the molecular basis for its actions hinders its translational development in to clinical use. Further research in to the basic mechanisms of TNFα secretion and action within the breast is required before we are able to consider it as a drug development target.

Footnotes

Acknowledgments

S.T. is supported by an Australian Postgraduate Award. K.C.K. is supported by a U.S. Department of Defense Postdoctoral Training Award (W81XWH-08-BCRP-POSTDOC). This work was supported by the National Health and Medical Research Council of Australia through a fellowship to CDC (#338518); the Victoria Breast Cancer Research Consortium Inc.; and the Victorian Government's Operational Infrastructure Support Program. PHI data audit #12–20.

Author Disclosure Statement

The authors declare that no competing financial interests exist.

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