Tumoral BRD4 expression in lymph node-negative breast cancer: association with T-bet+ tumor-infiltrating lymphocytes and disease-free survival

BRD4 (Bromodomain-Containing Protein 4) is a transcriptional epigenetic regulator that plays a crucial role in cancer and inflammatory diseases [1]. It is a member of the BET (Bromodomain and Extra Terminal domain) family that utilizes tandem bromodomains to recognize specific acetylated lysine residues in the N-terminal tails of histone proteins [2]. Upon interaction with chromatin, BRD4 has been shown to promote acetylation-dependent assembly of transcriptional regulator complexes that activate various transcriptional programs, such as those involved in cell proliferation and cell cycle control [3, 4].

Small molecule inhibitors that specifically target BET proteins have been demonstrated to interfere with expression of genes involved in cell growth and apoptosis evasion. Therapeutic benefits of the BET inhibitors have been observed in B-cell lymphoma [5] and acute myeloid leukemia [6, 7], as well as in lung [8], prostate [9], pancreatic [10], colorectal [11] and breast cancers [12]. Interestingly, BET inhibitors have also been shown to have an anti-inflammatory effect in the treatment of various inflammatory diseases and cancer [1, 13, 14], suggesting that BRD4 may have an active role in supporting inflammation.

Numerous studies have shown BRD4 to be important in the promotion of NF-kB-mediated transcription of inflammatory genes [15‐17], whose functions in cancer initiation and progression have shown to be manifold and complex [18, 19]. Considering the clinical benefits of cancer immunotherapies that have been demonstrated through blockades of immune inhibitory pathways and stimulation of immune effector functions in tumors, investigating the potential link between BRD4 and immune infiltration in cancer may present a novel insight into the regulatory role of BRD4 in tumor immune surveillance.

Breast cancer is a complex and heterogeneous disease. Despite improvements in disease classification using tumor-related prognostic markers, a large disparity of clinical outcomes continues to be seen. This reflects the limitation of utilizing intrinsic tumoral characteristics as the sole determining factors of disease progression. An increasing number of studies have demonstrated that the components of tumor microenvironment, including immune infiltration, interact dynamically with the tumor, and influence clinical outcome. Particularly, infiltration by T lymphocytes has been shown to be associated with a good prognosis in breast cancer patients, and higher response rate to neoadjuvant therapy [20‐27].

In two independent cohorts of women with familial breast cancer [28] and axillary node-negative (ANN) breast cancer [29], we have observed that T-bet+ tumor-infiltrating T lymphocytes (T-bet+ TILs) were associated with adverse clinicopathological features such as large tumor size, high grade, mutant p53, ER negativity, CK5 positivity, EGFR positivity, and basal molecular subtype [29, 30]. Despite being associated with an aggressive tumor phenotype, patients with a high level of T-bet+ TILs in their tumors had a favorable clinical outcome [29, 30]. T-bet is an immune-specific member of the T box family of transcription factors that is essential for differentiation of type 1 helper (Th1) T lymphocytes, as well as production of IFNy in CD4+ Th1 T lymphocytes and CD8+ cytotoxic T lymphocytes – subsets of immune cells that promote anti-tumoral inflammatory response [31, 32].

To examine how T-bet+ TILs may be associated with tumor development, we further investigated gene expression differences associated with T-bet+ TILs, and assessed their clinicopathological implications. Here we show that tumoral BRD4 expression is associated with T-bet+ TILs, relatively aggressive clinicopathological features, and a poor disease-free outcome in breast cancer.

In this prospectively accrued cohort of women with ANN breast cancer, we examined the relationship between BRD4 and T-bet+ TILs, and evaluated associations of BRD4 expression with Jagged1, clinicopathological features, and clinical outcomes.

We have demonstrated that BRD4 positivity (Allred score of 6 or higher) is significantly associated with T-bet+ TILs, which are a subset of T cells that we have previously determined to be associated with a good outcome in breast cancer patients, despite being associated with adverse clinicopathological features. This suggests a potential link between BRD4-associated tumor progression and the inflammatory lymphocytic infiltrate in breast tumors. BRD4 has been implicated in a number of studies for its role in promoting inflammation [13, 14, 41] notably via activating NF-kB-regulated pathways in cancer cells [17]. NF-kB is a major transcription factor involved in regulating immune and inflammatory responses, and in influencing cancer progression [42, 43]. In particular, NF-kB is crucial in mediating the synthesis of proinflammatory cytokines, such as TNF-α, IL-1, IL-6, and IL-8 [44], which suggests that BRD4 may be an upstream regulator of inflammatory immune response in tumors. Consequently, BRD4 inhibitors, such as JQ1 and I-BET, have been demonstrated to be effective suppressors of inflammation in treating various cancers and inflammatory diseases [13, 14, 41].

Furthermore, BRD4 was associated with pre-menopausal status, large tumor size, and high Ki-67 expression, which are characteristics that are generally associated with a basal subtype. Multiple studies have demonstrated that prognosis of basal breast cancer is positively associated with expression of immune response genes [45‐48]. Although no significant overall difference among intrinsic subtypes was observed between BRD4+/high and BRD4−/low tumors, the association of BRD4 expression with features related to the basal subtype reinforces the idea that the association of BRD4 with immunogenic tumors is potentially through its pro-inflammatory functions.

Women with T-bet−/low, BRD4+/high tumors had worse disease-free survival in comparison to the other women. One explanation may lay in the paradoxical roles of inflammation in cancer that is dependent on the immune composition of the tumor. The poor clinical outcome associated with the BRD4+/high group in the absence of T-bet+ TILs suggests that BRD4 may promote tumor progression through upregulation of chronic inflammatory pathways marked by the production of proinflammatory cytokines such as IL-1α, IL-1β, and IL-6. On the other hand, the relatively favorable outcome that is associated with T-bet+/high tumors despite having high BRD4 expression may indicate a dynamic immune interplay, in which the BRD4-mediated production of proinflammatory cytokines in the presence of tumor-specific T-bet+ TILs may reinforce an anti-tumor immune response. The context-specific role of inflammation in tumor development has been previously demonstrated in mouse models of myeloma and B-cell lymphoma [49]. In the latter study, increased local levels of both proinflammatory cytokines (IL-1α, IL-1β and IL-6) and Th1-associated cytokines (INFγ, IL-2 and IL-12) were shown to be consistently correlated with a successful tumor immune response mounted by tumor-specific CD4+ T cells. Hence, in a T-bet+ TIL-mediated tumor microenvironment, BRD4-mediated NF-kB activation, and subsequent proinflammatory cytokine production may contribute to tumor suppression as the pro-inflammatory cytokines have shown to be important in recruiting circulating leukocytes and activating CD4+ T cell functions.

Another explanation may lay in BRD4’s role in the upregulation of Jagged1 expression [2], which was observed to be associated with BRD4 positivity and T-bet+ TILs in this study. Jagged1 is one of the canonical ligands for the Notch receptor family [50, 51] that serves a multifaceted and highly context-dependent function in regular tissue development and cancer progression. The binding of Jagged1 to Notch1 or Notch3 receptors initiates their activation that involves proteolysis by γ-secretase and release of Notch intracellular domain (NICD). NICD translocates to the nucleus and associates with a transcription complex to regulate expression of target genes. In tumors, the paracrine Jagged1-Notch interaction between cancer cells has been shown to promote proliferation, epithelial-mesenchymal transition, angiogenesis, and metastasis [51]. A recent study demonstrated that BRD4 was the upstream regulator of Jagged1 expression and Notch1 signaling, and played an important role in sustaining breast cancer migration and invasion [2]. In patients, BRD4 and Jagged1 expression has been shown to correlate with the presence of distant metastases [2].

Based on the positive associations observed between BRD4 expression, Jagged1 expression, and T-bet+ TILs, Jagged1, through BRD4 regulation, may also be important in mediating tumor-immune cell interaction. Jagged1-mediated activation of Notch signaling has been shown to promote persistence of immature myeloid cells [52] and immunosuppressive IL-10 production [53], which are characteristics possessed by myeloid-deprived suppressor cells (MDSCs). A recent study by Sierra et al. has shown that humanized anti-Jagged1/2 suppressed tumor growth, decreased the accumulation and tolerogenic activity of MDSCs in tumors, and inhibited the expression of immunosuppressive factors, iNOS and arginase, which in turn, promoted CD8+ T cell infiltration into tumors, and improved the in vivo efficacy of T-cell based immunotherapy [54]. Hence, BRD4+/high tumors in the absence of T-bet+ TILs may exhibit BRD4-mediated upregulation of Jagged1 that may induce Jagged-1-Notch1-mediated accumulation and activation of MDSCs, and suppress the infiltration and anti-tumor activity of T-bet+ T cells.

In the presence of T-bet+ TILs, however, Jagged1 may promote anti-tumoral immune response as its expression has shown to be vital in co-stimulation and regulation of Th1 cells through binding of their cell surface receptor, CD46 (membrane cofactor protein, MCP) [55]. The latter study has shown that disturbance of Jagged1-CD46 crosstalk impeded IFNγ secretion in Th1 cells, and CD4+ T cells from patients with Jagged1 mutation (Alagille Syndrome) or CD46 deficiency failed to mount appropriate Th1 responses in vitro and in vivo. This finding, in addition to the positive association between Jagged1 and T-bet+ TILs observed in this study, suggests that in BRD4+/high, T-bet+/high tumors, BRD4-mediated upregulation of Jagged1 may reinforce the anti-tumoral activity of T-bet+ TILs, and facilitate disease-free survival of patients with breast cancer.

Tumoral BRD4 expression in breast cancer is significantly associated with T-bet+ TILs, clinicopathological features, and a poor disease-free survival in the absence of T-bet+ TILs. On the other hand, the favorable clinical outcome associated with BRD4 expression in tumors with high levels of T-bet+ TILs may reinforce the T-bet+ TIL-driven tumor immune surveillance. The context-specific association of BRD4 expression with disease-free survival based on the presence of T-bet+ TILs suggests that while the anti-inflammatory treatments against cancer, such as BET inhibitors, may be beneficial in reducing chronic inflammation, they may also reduce the tumor-suppressive, T-bet+ TIL-mediated inflammatory immune response. Hence, deeper understanding of BRD4’s immune modulatory roles in different immune contexts may be important in accurately administering BET inhibitors to patients without the risk of dampening the ongoing anti-tumor immune response.