1. Introduction
Exploring molecular changes in cancer has grown into an insightful technique in preclinical drug development trials. Cancers that are both estrogen-negative (ER-negative), progesterone-negative (PR-negative) and Her2-negative are considered basal-like, triple-negative breast cancers (TNBC). TNBC patients are considered more prone to reoccurrence and have the worst prognosis compared to other subtypes [
1]. Drug development assessments rely heavily on highly specific chemical biomarkers to increase the chances of efficacy. Recently, molecular efforts have revealed several possible therapy targets, including DNA repair markers, such as polyadenosine and ribose polymerase inhibitors (PARPi), the programmed cell death receptor (PD-L1), the epidermal growth factor (EGRF), the androgen pathway, and the NOTCH pathway [
2,
3], to combat the sustained progression of these aggressive subtypes. Chemotherapy and radiation therapy in combination with immunotherapy has become a promising approach for aggressive cases of cancers. In recent years, cancer mortality rates have slowly declined due to these remarkable advancements in molecular and clinical efforts. Despite these efforts, basal-like TNBC still occurs in 10–15% of patients, accounting for almost half of all breast cancer deaths, and is considered a highly aggressive and metastatic phenotype [
4].
Adipokines, chemokines, and pro-inflammatory factors all play a major role in triggering innate and adaptive immune responses. Surgery, chemotherapy, radiotherapy, and target therapy are considered the major forms of anticancer treatments, but recent advances have increased the efficacy of therapy options with immunotherapy in combination with conventional therapies [
5]. Enhancing antitumor adaptive immunity responses in immune-based therapy treatments has become a focus of preclinical studies. Adaptive immunity activation depends heavily on downstream innate responses that lead to pro-inflammatory IFN signaling, which should ultimately result in the elimination of pathogens. Immunity dysfunctions have been reported in many cancer patients, specifically breast cancer, melanoma, and gastrointestinal cancer, which usually results in immune evasion and, furthermore, failure of immunotherapy [
6]. Pro-inflammatory responses of adaptive immunity signaling must be explored in relation to excess adipocyte secretions. Mouse- and fibroblast-derived adipose tissue cells have been explored for their adipose-like phenotypical properties in biological studies. Adipocyte-conditioned media studies give clear outlines on the characteristics of secretory factors excreted by adipocytes [
7]. There has been promising evidence that adipocyte secretions trigger innate immune sensing in breast cancer [
8]. It is also widely understood that macrophage polarization is specific to secretions from components in the microenvironment [
9]. Cancer-associated adipocytes control several signals related to cancer progression and could influence immunosuppression and immune therapy evasion. This biological phenomenon is still yet to be fully explored.
During inflammation, there are a host of regulatory agents that facilitate downstream biological mechanics. The acute phase protein (APP), serum amyloid A (SAA), is a highly conserved apolipoprotein associated with high-density lipoproteins (HDLs). Under inflammatory conditions, SAA accumulation facilitates the change from anti-inflammatory HDL to pro-inflammatory HDL. Studies have reported its involvement in inflammatory-related diseases, such as atherosclerosis and rheumatoid arthritis, apparently through the control of NF-κB, JNK, C/EBP, Erk/MAPK, and PI3k/Akt/mTor signaling pathways [
10]. Emerging studies have also pointed towards its involvement in inflammatory-predisposed malignancies, such as breast, pancreatic, ovarian, liver, prostate, and renal carcinomas [
11]. Implications in cell migration, stimulating angiogenesis, and augmenting cytokine and chemokine expression, give hope for a potential molecular driver in some cancers. Interferon (IFN) and lipopolysaccharides (LPS) have been known to activate various classical genetic pathways in macrophages [
9]. Monocytic macrophages have been separated into four isoforms (M1–M4) that are broken down further based on specialized functionality. These four forms of polarization have been broken down even further to categorize their host of functions. Specifically, M2 macrophages acquire special characteristics that create immunosuppressive signaling via IL6, VISTA, IL10, and ROS expression [
12]. There have also been reports on macrophages being major binding factors of SAA in the injured liver. Further differential and polarization analysis shows evidence of SAA inducing M2b-like polarization rather than M2a or M2c subtypes [
11]. M2b macrophages show high IL10 and low IL12 production and are considered the more immunosuppressive isoform.
The immune suppressive potencies of M2 macrophages are found not only in cancer, but also in other diseases associated with chronic inflammation and autoimmune disorders. It is widely understood, but not fully examined, that cancer-associated adipocytes secrete factors that are associated with tumor progression and immune signaling [
13,
14]. In this study, basal-like, triple-negative breast cancer (TNBC) cell lines (MDA MB 231 and MDA MB 468) were grown in human visceral adipocyte-conditioned media (ACM) and analyzed by full transcriptome RNA sequencing. We showed the influence of ACM on key immunomodulators prompting myeloid-derived suppressor cell mimicry and T-cell inhibition. ACM also significantly upregulates the expression of genes associated with further driving tumor progression. Similarly, we showed that the acute-phase protein SAA1 is significantly upregulated in obese breast-cancer patients as well as in non-cancerous breast stroma samples. In contrast, the V-set domain member VTCN1, a key T-cell inhibitor, only appears in samples from obese patients with basal-like tumors. Specifically, obesity was shown to enhance immunosuppressive genes that prime the tumor microenvironment for macrophage infiltration and inactivity. Our findings also suggest that obese patients with basal-like breast cancer are more vulnerable to immunosuppressive signatures.
4. Discussion
Our findings give clear evidence of obese breast-tissue samples being more inclined to immunomodulatory gene expressions compared to those from non-obese patients. There was a significant upregulation of SAA1, IL10, and IL6R. SAA is an apolipoprotein of the high-density lipoprotein predominantly produced by the liver [
16]. SAA is a highly conserved protein known as a uremic toxin and has been found to induce intracellular immune defense macrophage-recruiting mechanisms. It is also known that patients suffering from septic conditions exhibit elevated SAA plasma levels. The role of SAA in acute and chronic inflammation disorders has not been fully explored. Wang et al. found SAA to be a major binding factor of M2 macrophages in an injured liver. Polarized M2 macrophages (IL10+, IL12-) give rise to a host of immunosuppressive responses in the TME [
17]. These macrophages, with more affinity to SAA, also produce IL1, IL6, and TNFA. It was reported that SAA1 induces the expression of CCL3, CCL2, and CXCL8 in monocytes. Gene expression analysis of non-tumorous breast stroma shows significantly high levels of IL10, IL6, and CCL2, key immunomodulators in humans (
Table 1). The macrophage recruitment agents S100a8 and S100a9, were also highly expressed in obese patients. These gene signatures are considered immunosuppressive due to their involvement in monocytic trafficking and T-cell inhibition [
18].
Furthermore, obese African-Americans showed a slight increase in specific gene expressions indicative of interferon signaling compared to European-Americans (
Figure 1). The expression of IFNG in breast cancer patients has some inconsistent data with respect to prognosis. These data give indications that interferon signaling in obese breast cancer patients may be dependent on ethnicity. Nearly 50% of patients in this dataset with poorly differentiated cases were obese (
Table 2). Luminal A was the most common subtype across all variables of BMI. Basal-like obese patients were the second most common subtype of all variables of BMI (
Table 2). Interferon signaling gene sets were significantly elevated in both Hallmark and Reactome data for African-American obese patients compared to European-American obese patients. There were also significantly enriched gene sets that are related to cytokine signaling and adaptive immunity in African-American obese breast-cancer patients compared to obese European-American patients (
Table 3).
Differentially expressed genes of basal-like obese patients compared to obese patients suffering from Luminal B and HER2+ subtypes were confirmed. Obesity significantly influenced immunomodulators in basal-like obese patients (BOB) compared to other subtypes (
Table 4). The degree of mRNA and protein sequence homology between SAA 1 and SAA 2 make them difficult to be distinguished. The synthesis of these isoforms relies heavily on inflammatory conditioning. Re-establishing homeostasis in acute-injury phase conditions calls for the liver to produce APPs like SAA. The biological role of SAA1 and its homologous counterpart SAA2 in in vivo and in vitro leukocyte-recruiting has been linked to the activation of toll-like receptors (TLR) 2 and 4. These pattern recognition receptors are key intrinsic cytokine regulators and lead to a host of innate immune responses. TLR2 activation via SAA1 recombinantly expressed in
Escherichia coli (
E. coli) has been recently linked to bacterial lipoproteins. Furthermore, Immunol et al. showed that the direct activation of monocytes was dependent upon SAA1 expression without chemokine interference. However, CCL3 and CCL8 expression in monocytes rely heavily on the expression of SAA1. Obese patients with basal-like subtypes (BOB) were analyzed for their upregulated gene expressions compared to other subtypes. There was a significant upregulation of CCL8, hrSAA 1 and 2, and VTCN1. These signaling molecules are associated with immune suppression. Microarray data also showed the influence of obesity on the upregulation of STAT1, which plays a major role in interferon signaling. Obesity was also associated with significantly upregulated expression of IL6 in BOB patients. This indicates the immune signaling capabilities of obese basal-like breast cancer patients, with IL6 being a key immune response molecule.
The predisposition to inflammation for TNBC patients increases serum amyloid levels and mediates cancer progression and high mortality. Notably, high levels of VTCN1 were shown in MDA MB 231 ACM samples. This is a key regulator of T-cell activities. There was a slightly different story for the MDA MB 468 RNA sequencing data. Molecular changes influenced by ACM were more closely related to interferon signaling. Like the GSEA of patient data, there were key interferon signaling genes being upregulated in the presence of excess adiposity in association with the African-American basal-like cell line (MDA MB 468). Genes such as JAK3, STAT3, and STAT1 were significantly upregulated in MDA MB 468 adipocyte-conditioned media samples compared to control (
Table 6). Additionally, the tumor suppressive genes SAA1, VSIR, and BCL3 were also significantly upregulated in ACM samples. These genes are proven to be immunosuppressive in that they promote the inactivation of key immune cells.