NF-κB as the main node of resistance to receptor tyrosine kinase inhibitors in triple-negative breast cancer

Abstract

Graphical abstract

Although accounting for merely a minute portion of diagnosed breast cancers, disproportionate number of deaths and associated low survival rate of patients have made triple-negative breast cancer to be considered as the most lethal breast cancer subtype. More importantly, intrinsic or developed resistance to chemotherapeutic regimens and disappointing outcomes of trials associated with many newly developed agents are other obstacles in establishment of a durable response in these patients. Interestingly, these happen despite the outstanding preclinical outcomes observed by these agents, most importantly among them, targeted receptor tyrosine kinase inhibitors. Pursuing these disappointing outcomes, especially in the case of targeted receptor tyrosine kinase inhibitors, many researches have focused on identification of the hidden factors involved. Highly inflammatory, rich in reactive oxygen species, and hypoxic microenvironment of triple-negative breast cancer tumors and the involving mediators were the first suggestions for observed resistance and poor clinical outcomes of targeted receptor tyrosine kinase inhibitors. Interestingly, for all aberrantly expressed mediators observed in microenvironment, downstream pathways converge in a common node, nothing but the nuclear factor-κB, the insidious factor proposed to be the cause of many events opposing achievement of a desired outcome. In first section of current review, we describe the signaling pathways underlying activation of receptor tyrosine kinases and their convergence at the nuclear factor-κB node, and in next section, we demonstrate how unique hypoxic, inflammatory, rich in free-radical microenvironment of triple-negative breast cancer exacerbate pathways in which otherwise could become mostly suppressed by receptor tyrosine kinase inhibitors.

Introduction

Based on immunohistochemical classification of breast cancers, triple-negative breast cancer (TNBC) is defined as the specific subtype of epithelial breast tumors, negative for expressing estrogen receptor (ER), progesterone receptor (PR) and overexpressed HER2, consisting about 10%–14% of all breast cancers. ¹ TNBC tumors are mostly characterized by high mitotic rate, enhanced lymphocytic infiltration, high grade, and large size. Brain metastasis takes place in about 15% of TNBC cases and is associated with very poor prognosis and significant shortened survival time. ² Occurrence of brain metastasis years after complete resection of breast tumor strongly suggest that disseminated TNBC cells possess particular characteristics which allow them to take over this organ even at the absence of primary tumor. ³ Interestingly, this usually happens in spite of desirable responses of patients with TNBC tumors to initial chemotherapy regimens. Contrary to other tumors, no certain chemotherapy protocol has been established for TNBC yet. Instead, the therapeutic regimen is mostly planned based on tumors’ characteristics including size, number of involved lymph nodes and existing co-morbidities. Absence of ER/PR/HER2 overexpression pattern has made endocrine therapy with tamoxifen, aromatase inhibitors and HER2 directed therapies useless for TNBC. Furthermore, TNBC is mostly resistance to anthracyclines and taxans, making the number of potent therapeutic agents more restricted. ⁴ Recent studies have clearly demonstrated that receptor tyrosine kinases (RTKs) are mostly overexpressed in TNBC cells. These receptors demonstrate high affinity for several specific polypeptides including growth factors, cytokines and hormones. Since the first discovery of RTKs about 25 years ago, multiple members of these cell surface receptors have been identified and shown to play key roles in regulation of essential cellular processes, including proliferation, differentiation, cell survival, migration and cell cycle control. ⁵ All defined RTKs have a common molecular scaffold, consisting of a ligand-binding region located in extracellular domain, a single transmembrane helix, and a cytoplasmic region containing the protein tyrosine kinase (TK) domain together with carboxy terminal and juxta membrane regulatory regions. ⁶ Epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and insulin-like growth factor receptor (IGF1-R) are among the most important RTKs presented in TNBC cells. Nevertheless, monotherapy with their selective inhibitors including trastuzomab, bevacizumab, or imatinib did not demonstrate any significant improvements or lowering in progression rate of TNBCs. ⁷ Consequently, precise interpretation of underlying mechanisms consistent with these observations appears to be essential for developing new generation of more effective inhibitors or co-administration of other agents for achieving optimum results.

Enhanced expression of acute phase proteins (APPs) has shown to increase the recurrence risk by two-folds in TNBC. As specific biomarkers, enhancement in expression of these proteins mostly results from overexpression of inflammatory mediators specially the pro-inflammatory cytokines.^8,9 Additionally, several studies have demonstrated a significant relation between overexpression of interleukin (IL)-6 and poor prognosis in TNBC patients.^10–12 From other side, it has been shown that long-term consumption of non-steroidal anti-inflammatory drugs (NSAIDs) is together with significant reduced risk of breast cancer development. These data all suggest a significant relation between inflammation and development of breast cancer. Furthermore, growing body of evidence demonstrates that pro-inflammatory cytokines including IL-6 and IL-8 enhance tumor growth and metastasis through alteration of tumor cell biology and triggering activation of stromal cells including vascular endothelial cells, tumor-initiated macrophages, and fibroblasts in microenvironment of TNBC.^13–15

As the prototypical transcription factor involved in modulation of inflammatory response, the nuclear factor-κB (NF-κB) has mostly been involved in the expression of several pro-inflammatory proteins including cytokines, chemokines, and adhesion molecules. Tumors can enhance NF-κB activity through increasing cytokine release from stromal cells and fibroblasts in tumor microenvironment. Overexpression of NF-κB has largely been manifested in TNBC,^8,16 and since it is the converging point of several signaling pathways transduced by RTKs, one can suggest that overexpression of NF-κB can be considered as one of the main reasons for RTK inhibitors not inducing desired responses in clinical trials. Furthermore, several external stimulators such as reactive oxygen species (ROS) and hypoxy-induced factor (HIF) can promote activation of NF-κB, and since these mediators have long been discovered as chemoresistance-inducing factors, the probability of NF-κB overexpression involvement in resistance development to RTK inhibitors becomes more probable. ¹⁷ Mentioning this point also seems critical that pro-inflammatory cytokines, including IL-6 and IL-8, significantly develop crosstalks with both RTKs and NF-κB. ¹⁸ This may be the answer to the question that why despite the administration of RTK inhibitors we observe resistance in chemotherapy. Based on this brief preface, here, first we introduce signaling pathways transduced through RTK receptors activation and then investigate the crosstalk of signaling pathways initiated by RTKs with pro-inflammatory IL-6 and IL-8 and NF-κB and finally describe how internal and external stimulators increase the activity of NF-κB.

Signaling pathways transduced by RTKs activating ligands

Ras/Raf/extracellular signal–regulated protein kinase pathway

The Ras/Raf/mitogen-activated protein kinase (MAPK) pathway is probably the most well-known signaling cascade involved in controlling cell growth, proliferation, and differentiation. For instance, it has been elicited that this pathway by controlling integrin-mediated signaling pathway and consequently cell migration capacity can significantly affect tumor metastasis.^19,20 Furthermore, by influencing angiogenesis and neovascularization, Ras pathway can also affect tumor propagation. ²¹ Based on the literature, Ras is the most frequently mutated oncogene reported in TNBC ²² and even in the absence of stimulating ligand, Ras can initiate oncogenesis through amplification of related downstream genes and activation of several upstream pathways. Ras has shown to be significantly correlated with HER2/neu receptor too. It has been shown that Ras superfamily cognate genes can encode several small guanosine triphosphate (GTP)-binding proteins which can in turn regenerate multiple cellular processes including membrane trafficking, organization of cytoskeleton, and several processes involved in cell differentiation. ²³ So far, three members of Ras family oncogenes have been identified, including H-Ras, K-Ras, and N-Ras. ²⁴ Among them, K-Ras is the most important one in TNBC. ²⁴ K-Ras is synthesized as an inactive cytosolic pro-peptide which becomes localized in cell membrane after undergoing several modifications in carboxyl terminus, converting it to a more hydrophobic agent. There, it frequently cycles between inactive guanosine di-phosphate-bound and active GTP-bound state and takes part as an initiator for multiple effector kinases including Raf and MAPK. ²⁵ In the case of mutation or overactivation by other pathways, Ras favors its GTP-binding state which in turn more frequently activates downstream effectors. This can even take place in the absence of primary cognate ligands such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), granting a proliferative state for tumors. Ligand coupling to EGFR results in oligomerization of receptors and juxtaposition of receptors’ cytoplasmic domains in a way to trigger kinase activity and subsequent phosphorylation. ⁶ Then, growth factor receptor bound protein 2 (Grb2) becomes capable of recognizing sequence homology 2 (SH2) domains and further recruits guanine nucleotide exchange factors (GEFs) such as cell division cycle 25 (CDC 25) and SOS1 to the cell membrane. There, GEFs interact with Ras and induce conformational change and further exchange of guanosine diphosphate (GDP) with GTP, resulting in subsequent activation of Ras. Following this step, Raf recruits to the cell membrane through lipid binding and couples with Ras switch 1 domain. ²⁶ This coupling phosphorylates MAPK and further activates downstream signaling cascades. Activated MAPK in next step phosphorylates extracellular signal–regulated protein kinases 1 and 2 (ERK1/2) which in turn continues activation of a vast variety of nuclear transcription factors and other kinases including MNK1, MNK2, Ets2, as well as Tob, a well-known anti-proliferative protein. Most of the mentioned proteins are involved in activation of cell transformation. As a serine/threonine kinase, MAPK promotes both cell survival and apoptosis through engaging several mediators including stress-activated protein kinase (SAPK) and NF-κB. ²⁷ More importantly, K-Ras can also activate other downstream K-Ras effectors including PI3K cell survival pathway, small GTP-binding proteins including Rac, Rho, and also the SAPK pathway c-Jun N-terminal kinase (JNK) pathway. ²⁷ It has also been shown that activation of K-Ras in response to cytokine stimulation results in activation of dual-specificity p38^MAPK kinase (MKK3 and MKK6) which in turn result in phosphorylation of p38^MAPK and JNK kinase (MKK4 and MKK7) further phosphorylating JNK. As mentioned earlier, the most important activators of Ras pathway are EGFR and PDGFR. Nevertheless, activation of many other receptors including pro-inflammatory cytokine receptors is also important in activation of K-Ras pathway too. ²⁸ As a result, aberrant activation of K-Ras through overexpression of pro-inflammatory cytokines can significantly result in the development of resistance to EGFR inhibitors including cetoximab, gefitinib, erlotinib, and lapatinib.

PI3K/AKT/mammalian target of rapamycin pathways

Performed studies on TNBC cells have shown that PI3K/AKT/mammalian target of rapamycin (mTOR) is an important pathway involved in metabolism, growth, and survival of these cancerous cells. ²⁹ This pathway can be initiated either by activation of G-protein coupled receptors (GPCRs) or RTKs, including EGFR and FGFR. ³⁰ PI3K initiates pathway by phosphorylation of phosphatidyl inositol diphosphate (PIP2) and formation of PIP3 which in turn contributes to the activation of an oncogene named Akt. The main inhibitor of this pathway has proposed to be the tumor suppressor gene phosphatase and tensin homology (PTEN) which acts as a dephosphorylating agent, converting PIP3 to PIP2. Another suppressor of this pathway which has been discovered recently is a lipid phosphatase, known as Inositol polyphosphatase 4-phosphatase type II (INPP4B). As another serine/threonine protein kinase, Akt phosphorylates several mediators involved in cell survival, proliferation and metabolic pathways; ³¹ most importantly, tuberous sclerosis complex 2 (TSC2), which in turn, results in activation of mTOR. mTOR itself enhances protein synthesis through activating eukaryotic translation initiation factor elf4E binding protein 1 (4EBP1) and p70S6 kinase (S6K). Consequently, activation of Akt results in significant interrelation between cell growth, metabolism, and apoptosis. As a serine/threonine kinase, mTOR is composed of mTORC1 and mTORC2 protein complexes. So far, discovered inhibitors of mTOR are mostly preferential for mTORC1. However, inhibition of both complexes is critical for controlling cancer propagation. Selective inhibition of mTORC1 results in negative feedback on PI3K pathway by involvement of S6K1 and IRS1 and can enhance cell growth through reflective activation of Akt through mTORC2. ³² As PI3K pathway is critical for maintaining normal homeostasis of glucose, protein synthesis, and metabolism and from other side since insulin and IGF-1 demonstrate their activities through similar pathway, a significant correlation exist between metabolism deregulation and oncogenesis. ³³ Studies have shown that overexpression of several upstream activators including HER2 and IGF-1, loss of PTEN, and more importantly activation of pro-inflammatory cytokines including IL-6 and IL-8 can result in hyper-activation of PI3K and enhance cancer propagation. ³⁴

JAK/signal transducer and activator of transcription pathway and cytokines

Signal transducers and activators of transcription (STATs) are specific group of transcription factors playing key role in cancer propagation. ³⁵ Considering normal cells, STAT3 activation is mostly transient and is tightly regulated both in extent and duration. Contrarily, in several cancers, STAT3 becomes constitutively activated and leads to continuous activation and expression of target genes involved in invasion and angiogenesis. The main activator of STAT3 in malignancies is aberrant production of IL-6 which specifically signals via a complex of IL-6 receptor alpha (IL-6α) and gp130 to promote activation of Janus kinases (JAKs) and further induction of tyrosine phosphorylation of STAT3. This type of activation of STAT3 has been identified in multiple types of tumors including breast, colon, myeloma, head and neck and melanoma.^36,37 As STATs are not chemical oncoproteins themselves, tumorigenic potency of this pathway was mostly underestimated initially. Recently it has been demonstrated that constitutive activation of JAK can result in tumorigenesis at least by two ways: first by continuous activation of downstream STAT family proteins which are potent transcription activators and second through a global counteracting heterochromatic gene silencing activity by means of non-STAT cognate gene depression.^38,39 Both mechanisms act in a way to help STAT coactivators (e.g. CBP/p300) acetylate surrounding histone proteins more effectively and cause chromatin changes, enabling further expression of STAT-independent genes. ³⁸ As mentioned earlier, STATs themselves are not considered as typical oncoproteins; nevertheless, continuous signaling through STATs is frequently observed during cancer. This has also been reported that STAT3 is the main player in the signaling pathway involved in breast cancer propagation.^40–42 STAT is mainly activated by several cytokine receptors, especially the ones containing gp/30 chains such as IL-6. ⁴³ In this pathway, STAT involves in suppression of apoptosis and stimulation of cell proliferation, the one which is mostly observed during chronic inflammation. Parallel to this, STAT cognate genes, including Bcl-XL, a well-known apoptosis suppressor and C-Myc an important cell proliferation inducer are also overexpressed. ⁴¹ Since STAT3 is also an important mediator involved in chronic inflammation, a significant risk exists between chronic inflammation and induction of cancer. ⁴² As mentioned earlier, this pathway can also become activated by VEGFR ligation. Consequently, cytokines can result in development of resistance to VEGFR inhibitors including bevacizumab, sunitinib, and apatinib by overactivation of this signaling pathway. Resistance to PDGFR inhibitors can also develop by overexpression of this pathway, some of which includes imatinib, sunitinib, and sorafenib.

So far, in mammals, four types of JAKs and seven types of STAT proteins have been identified. Based on the structure of intracellular domain of these receptor, each cytokine can activate a unique set of individual JAK and STAT. ⁴⁴ Cytokines coupling with their cognate transmembrane receptors result in dimerization and/or reorganization of dimerized receptors ⁴⁵ which in turn cross activates JAKs. In next step, JAKs phosphorylate tyrosine residues adjacent to the c-Terminus and result in STAT protein detachment from receptor, formation of homo- or hetero-dimers, transportation toward nucleus, and induction of related gene transcription. Commonly, STAT cognate genes mostly involve in intracellular regulatory pathways of cell proliferation such as cytokine production or cell survival including anti-apoptotic Bcl family proteins.^46,47 Furthermore, they are capable of secreting mediators capable of enhancing tumor formation and propagation, linked with JAK/STAT activation.

The crosstalk between PI3K/Akt/mTOR and Ras/Raf/MAPK pathways

As depicted in Figure 1, based on literature and reported studies, it has been shown that a significant correlation exists between the specific kinases of these pathways in a way that decrease in one results in enhancement of the other one and consequently, signals for cell survival will be transmitted untouched and with high fidelity.^48,49 Several studies have demonstrated that PI3K can result in activation of Ras. ⁵⁰ For instance, AKT can restrict Rafs phosphorylation, which in turn reduces MAPK signaling. ⁵¹ Furthermore, Ras can result in suppression of PTEN expression. ⁵² As both of these pathways result in activation of mTORC1 and 4EBP1, it has been proposed that loss of the inhibitory feedback resulted from suppressing Raf/MEK/ERK pathway leads to increased activity of PI3K/Akt/mTOR pathway. This in part can explain why soon after administration of RTK inhibitors, resistance development occurs. Additionally, it has been shown that inhibition of MEK can also result in significant increase in Akt expression, further proving the proposed loop. ⁵³ Additionally, PI3K inhibition has also demonstrated to result in a significant increase in MAPK/ERK pathway. ³⁴ the consequence of these crosstalks is the main cause of resistance development to c-MET pathway inhibitors such as Tivantinib and Crizotinib. ⁵⁴

Figure 1.

The crosstalk between Ras/Raf/MAPK and PI3K/Akt/mTOR signaling pathways. Both signaling pathways end in activation of mTORC1, responsible in regulation of cell survival, proliferation, and protein translation. PI3K can induce activation of Ras which in turn suppresses expression of PTEN. From other side, AKT restricts phosphorylation of Raf which in turn can suppress MAPK signaling pathway. Additionally, inhibition of MEK by PDK1 results in increased activation of AKT and ERK which can further inhibit TSC1 and TSC2. The classical regulatory S6-IRS1-PI3K feedback loop also leads to PI3K and ERK signaling pathway activation.

Involvement of inflammation in RTK inhibitors: NF-κB the main node of crosstalk

Activation of Akt plays an important role in the regulation of several downstream targets including mTOR, GSK3β, and NF-κB all of which are important modulators of different cellular functions including cell survival, metabolism, cycling, protein expression, and motility. Of these, NF-κB is presented in an inactive form coupled with inhibitor kappa B (IκB) protein in cytoplasm. ⁵⁵ PI3K/Akt activated pathway with either internal or external factors initiate IκB kinase (IKK)α activation and through this induce phosphorylation of IκB proteins. Phosphorylated IκB undergoes proteasomal degradation and cause activation of NF-κB and subsequent initiation of several proteins’ transcription. ⁵⁶ The activation of MAPK K-Ras can also induce activation of NF-κB. Based on these pathways, the most important outcome of RTKs activation is activation of NF-κB pathway. Initially, discovered as inflammatory regulatory factor, it has now been understood that NF-κB takes part in different steps of tumor development, proliferation, differentiation, and more importantly epithelial–mesenchymal transition (EMT) and induction of metastasis. In the case of TNBC, NF-κB plays an important role in cells survival and proliferation has been significantly identified. ¹⁸

Transcription factors can exert reciprocal effects with each other through verity of methods. For instance, they can physically associate with each other and cause changes in transcriptional activities and DNA binding. STAT3, Smad 3 and Smad 4, AFT3 and ERs can directly couple with NF-κB. Additionally, transcription factors can couple with each other in proximity through their cognate sequences of promoters and facilitate recruitment of several components essential for transcriptional machinery and as a result either facilitate or suppress effects of each other. ⁵⁷ As discussed above, NF-κB and STAT3 can regulate expression of several genes involved in anti-apoptosis and good cycle control in a cooperative manner. This must be also taken in mind that the JAK/STAT pathway is mostly upregulated through interactions of VEGF or pro-inflammatory factors with their consistent receptors in TNBC.

NF-κB and STAT cooperation can further control transcription of genes involved in cytokines and chemokines production. As reported by Hartman et al., examining TNBC, inflammatory-related genes are produced through ER-negative genes, most of which are necessary for anchorage-independent growth of TNBC cells. Cytokines responsible for anchorage-independent growth of cells are mostly synthetized by activation of NF-κB transcription factor. Furthermore, evidence exists that IL-6 and IL-8 autocrine production takes place in immune and other stromal cells which in turn react in a paracrine manner resulting in enhanced neovascularization and inflammation-dependent carcinogenesis. ¹¹

Studies have demonstrated that p65 and p50 NF-κB can physically interact with STAT3 and contribute to the recruitment of NF-κB to STAT3 promoters. Furthermore, recruitment of acetyl transferase p300 and acetylation of NF-κB through modification of RelA prolongs retention of NF-κB in nucleus, converting its physiological role into tumorigenic. NF-κB further enhances IL-6 production and result in maintenance of this positive loop, making situation more aggravated. Recent data have also proposed that mutant p35 can express p52 NF-κB through acetylating histone by means of CBP and STAT2 recruitment by means of its promoter CBP-mediated acetylation. ⁵⁸ Furthermore, negotiation between p53 and NF-κB has shown to induce maximum activity of NF-κB. ⁵⁹

NF-κB also develops several crosstalks with microRNAs (miRNAs). MiRNAs are group of small non-coding single-stranded RNAs which cleave messenger RNAs (mRNAs) through binding with their 3′-untranslated region (UTR). ⁶⁰ Due to the diversity of miRNAs and their cognate mRNAs, different signaling molecules and consequently their downstream pathways can be regulated by miRNAs. Additionally, miRNAs are transcriptional targets themselves and can downregulate gene expression through activation of corresponding transcription factors. As miRNAs are transcriptional targets themselves, they can downregulate gene expression through activation of corresponding transcriptional factors. MiR-9, miR-21, miR-143, miR-146, and miR-224 are some of the transcriptional targets of NF-κB.^61–63 Mentioned miRNAs mostly take part in feedback mechanisms regulating NF-κB activity through targeting expression of several upstream signaling proteins or the members of NF-κB family themselves. Nevertheless, NF-κB can also modulate expression of proteins involved in regulation of miRNAs. Most important examples include induction of Lin 28 expression, the protein mostly involved in inhibition of processing and maturation of Let7 miRNA family members. These miRNAs are mostly involved in tumor suppression. Let7-miRNAs also involve in suppression of IL-6 expression and consequently result in further synthesis and release of IL-6, resulting in earlier mentioned positive feedback loop. ⁶⁴ Recently, it has been discovered that loss of tumor suppressor miRNA miR-146b which is a target gene of STAT3 is associated with persistent activation of STAT3 in breast cancer. Xiang et al. discovered that methylation of miR-146b promoter in breast cancer is together with suppressed miR-146b expression which in turn results in NF-κB-mediated production of IL-6, activation of STAT3, and subsequent IL-6/STAT3-mediated migration and invasion of breast cancer cells through a negative feedback loop. Consistent with this finding, they demonstrated that higher expression of miR-146b positively correlated with breast cancer patient’s survival rate. Increased IL-6 expression and subsequent phosphorylation of STAT3 were also observed in these patients. ⁶⁵

Hypoxia inducible factor (HIF-1α), ROS, and NF-κB

HIF-1α is the most important factor involved in controlling cell response to hypoxia. It has been shown that hypoxia can significantly stabilize inducible α subunit of HIF-1α and consequently restrict further hydroxylation and degradation by proteasomes. Recent data suggest that HIF-1α can also respond to several stimuli during normoxic conditions, some of which include growth factors, IGF-1, ROS, and cytokines such as tumor necrosis factor (TNF)-α. Transcriptional regulation of HIF-1α by NF-κB which is also an important redox-sensitive transcription factor has been proposed to be the most important mechanism involved in this process. NF-κB specifically binds to the proximal promoter of HIF-1α gene and consequently regulates cancer cell response to inflammation, oxidative stress, and more importantly hypoxia. A significant crosstalk presents between NF-κB and HIF pathway in response to TNF-α and hepatocyte growth factor (HGF).^66,67 Subsequent activation of NF-κB through phosphorylation of IκB has significantly been correlated with levels of both HIF-1α mRNA and protein basal levels.^68,69 p50 and p65 subunits of NF-κB have been shown to directly react with HIF-1α promoter. ⁷⁰ Consequently, NF-κB and HIF-1α cooperatively modulate transcription of several genes responsible for regulation of several vital cellular processes including adaptation to hypoxia, reprogramming of metabolic response of cells in hypoxia, digestion of extracellular matrix of tumor cells, cell–cell adhesions, and so on. Additionally, discovery of the fact that alarmin receptor genes including RAGE, P2X7, and specific types of toll-like receptors (TLRs) are activated by HIF-1α and further links inflammation and hypoxia with tumor pathogenesis. ⁷¹ This is important since activation of alarmin receptors results in activation of NF-κB and subsequent expression of several pro-inflammatory cytokine. ⁷² Therefore, microenvironment takes part in tumor propagation mostly through activity of pro-inflammatory cytokines, growth factors, and generation of a microenvironment rich in ROS actors’ new mutations in cancerous cells and further development of RTK inhibitor chemotherapy agents (Figure 2).

Figure 2.

The crosstalk between hypoxia, reactive oxygen species, receptor tyrosine kinase, and NF-κB. HIF-α mostly involves in controlling cells response to hypoxia. Hypoxia enhances activation of PI3K/AKT/mTOR and MAPK/ERK pathways and transcription factor NF-κB, all of which result in enhancement of HIF-α synthesis. In turn, HIF-α activates expression of several cognate genes and results in enhancement of angiogenesis and blood vessel formation, metastasis, and modulation of apoptosis and cell metabolism.

Main consequences of NF-κB activation

Once activated, NF-κB initiates a complex gene response which in turn activates inflammatory and reparative responses. Most of these genes have also been involved in acquisition of malignant hallmarks by cells. Activation of NF-κB in leukocytes results in overexpression and production of several pro-inflammatory molecules including prostaglandins, leukotrienes, and nitric oxide (NO), resulting in amplification of inflammatory reparative response (IRR) and further tumor development. ⁷³ Molecules released by these cells also involve in neoangiogenesis, recruitment of leukocytes into tumor microenvironment, and also induction of EMT. ⁷⁴ Based on the type of involved cytokine, they can induce tumor proliferation (IL-2), polarization of tumor cells (Th1 cytokines), and expression of adhesive molecules. ⁷⁵ Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMs) are also produced in response to activation of NF-κB, disrupting MMP/TIM ratio, enhancing protease activity over basement membrane, and enhancing invasiveness of malignant tumors.^76,77 Activation of NF-κB can also enhance cell detachment through affecting localization of adhesion molecules including cadherins, intercellular adhesion molecules (ICAMs), selectins, and their counterparts in adherence junctions. ⁷⁸ VEGFs and their downstream-related genes are also the other cognate genes of NF-κB activation, pushing neoangiogenesis further in tumor site. ⁷⁹ These evidences significantly suggest that NF-κB is an important transcription factor involved in the development of resistance to chemotherapy and an important promoter of tumor propagation and invasive behavior of cancer cells.

Conclusion and future perspective

Establishment of an effective therapy for TNBC due to the restricted therapeutic options relative to the lack of ER/PR/HER2 overexpression and intrinsic or developed resistance still remains as a great challenge in breast cancer therapy. As discussed in this review, one of the most important concepts in targeted TNBC chemotherapy is consideration of overlapping pathways induced by cytokines, ROS, and hypoxia. Although overexpression of RTKs is a unique characteristic of TNBC cells, complex crosstalks between activated pathways make prediction of chemotherapy outcomes so difficult. In most cases, common converging point of different RTK activation is NF-κB; nevertheless, activation of other transcription factors must also be considered. As signaling pathways activated by ROS, hypoxia, and inflammation result in activation of NF-κB, inhibition of NF-κB caused by RTK inhibitors is mostly compensated and subsequently resistance develops. Consequently, even in the case of administering RTK inhibitors, still co-administration of agents reducing inflammation of tumor microenvironment, free radical scavenging agents, and HIF-1α inhibitors for achieving optimum benefits from targeted therapies appears to be critical. Furthermore, due to the crosstalk between Ras/Raf/MAPK and PI3K/AKT/mTOR pathways, administration of targeted agents with potency of inhibiting both pathways simultaneously seem to be more effective. Additionally, due to the crosstalk of RTKs downstream pathways with other receptors such as MUC-1C and several others which have yet to be discovered make condition more complex. Future studies appear to be more important in a way to develop new chemotherapy agents including multivalent bispecific antibodies with different signal transducing pathways appears to be a desirable choice instead of targeting just one pathway. Overall, considering one pathway for targeted therapy in TNBC by existing agents is sentenced to absolute failure either soon or late.