Research ArticleExperimental Studies
Open Access

Brain Metastases Are Regulated by Immuno-inflammatory Signaling Pathways Governed by STAT3, MAPK and Tumor Suppressor p53 Status: Possible Therapeutic Targets

SABRINA L. ZELLER, ERIS SPIROLLARI, ALIS J. DICPINIGAITIS, JOHN V. WAINWRIGHT, SIMON J. HANFT, CHIRAG D. GANDHI and MEENA JHANWAR-UNIYAL
Anticancer Research January 2024, 44 (1) 13-22; DOI: https://doi.org/10.21873/anticanres.16783

Abstract

Background/Aim: Brain metastasis (BM) is a complex multi-step process involving various immune checkpoint proteins. Mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1/2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3) are implicated in tumorigenesis and are critical upstream regulators of Programmed Death Ligand 1 (PD-L1), an immunotherapy target. Tumor suppressor p53, dysregulated in cancers, regulates STAT3 and ERK1/2 signaling. This study examined the roles of STAT3, MAPK and p53 status in BM initiation and maintenance. Materials and Methods: Twenty-six BM, with various primary malignancies, were used (IRB-approved) to determine mutant p53 (p53mt), pSTAT3Tyr705, pERK1/2Thr202/Tyr204, and PD-L1 expression using immunohistochemistry. cDNA microarray was used for gene expression analysis. Brain-metastatic breast cancer cells (MDA-MB-231) were treated with STAT3 (NSC74859) or MAPK/ERK1/2 (U0126) inhibitors in regular or astrocytic media. ERK1/2 pathway was assessed using western blotting, and cell proliferation and migration were determined using MTT and scratch-wound assays, respectively. Results: pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 were expressed at tumor margins, whereas p53mt and PD-L1 were uniformly expressed, with significant overlap between expression of these proteins. Gene expression analysis identified alterations in 18 p53- and 32 STAT3- or MAPK-associated genes contributing to dysregulated immune responses and cell cycle regulation. U0126 and NSC74859 reduced pERK1/2Thr202/Tyr204 expression. Cell proliferation decreased following each treatment (p≤0.01). Migration stagnated following U0126 treatment in astrocytic media (p≤0.01). Conclusion: Activation of STAT3 and ERK1/2 promotes BM and provides compelling evidence for use of STAT3, ERK1/2 and p53 status as potential immunotherapeutic targets in BM.

Key Words:

Brain metastases (BM) occur in nearly 20% of all patients diagnosed with cancer in the United States, affecting approximately 170,000 people per year (1). The most common primary sites of BM are lung (40%-50%), breast (15%-25%), and malignant melanoma (5%-20%) (2). Despite improved detection with technological advancements and prolonged survival in cancer patients overall, the incidence of BM continues to increase (3). Several studies have implicated the roles of specific genes associated with brain metastases (4, 5). BM is a complex multi-step process that involves epithelial-to-mesenchymal transition (EMT), a mechanism regulated by the mammalian target of rapamycin (mTOR) pathway by which metastatic cells establish foci of growth at distant sites (6, 7). The first phase of metastasis involves the migration of a cancer cell to a distant organ, followed by a second phase involving development of a metastatic lesion at the distant site (8). Following the “Soil and Seed” hypothesis, it appears that tumor cell proliferation vastly depends on the host tissue environment as well as the tumor metabolic microenvironment (9). Indeed, a recent study has shown that a crosstalk exists between lung cancer cells and astrocytes that leads to induction of a gene expression profile resembling that seen in neurons and astrocytes during brain development at early stages (10). Gene expression profiling of BM originating from lung adenocarcinoma revealed 1,561 gene alterations involved in establishing and promoting BM, with over-expression of genes associated with invasion and metastasis (phosphatase and tensin homolog, PTEN; matrix metalloproteinase-1, MMP-1), adhesion (integrin α3 and fibronectin-1), angiogenesis (vascular endothelial growth factor - VEGF), and cell migration (Rho GTPase) (5).

Alteration in the p53 gene has been shown to contribute to tumor initiation as well as metastasis. p53 gene alterations are frequent events in malignant disease, though the frequency of mutation in p53 gene differs significantly between tumor types; high proportions of mutations are found in lung cancer, breast cancer, lower gastrointestinal cancers, and glial brain tumors, while lower frequencies are reported in melanoma and sarcomas (11). Several p53 mutations identified in human cancer encode mutant proteins, which possess gain of function, such as the ability to cooperate with activated oncogenes to transform primary cells. The most common missense mutations in human cancer are termed ‘hotspot mutations’, and complex mutations like insertion/deletion/nonsense are also found in various tumors (12). Several studies have demonstrated the presence of specific p53 gene mutations in central nervous system (CNS) metastases of breast cancer (13). It is important to note that mutations in p53 gene found in CNS metastatic samples differ from the type of mutation present in matched primary tumors (14).

Signal transducer and activator of transcription 3 (STAT3) is an oncogene and multifaceted transcription factor shown to be involved in multiple cellular functions. The role of STAT3 anti-tumor immunity has recently been established. Activated (phosphorylated) STAT3 (pSTAT3Tyr705), which forms dimers and transports into the nucleus, represents a key transcription factor that critically controls proliferation, invasiveness, survival, and metastasis (15). It also modifies the immune response through various mechanisms, including regulation of programmed death ligand 1 (PD-L1) expression (16). Expression of pSTAT3Tyr705 was positively correlated with PD-L1 expression in breast cancer. In addition, knockdown of the Stat3 gene in a murine model of breast cancer expectedly resulted in decreased levels of PD-L1, thus producing decreased tumor volume, tumor weight, and a reduced pulmonary metastatic index (17).

The mitogen-activated protein kinase (MAPK) signaling pathways are often activated in cancers by genetic alterations in upstream pathways and have been implicated in the BM and EMT processes. The MAPK pathway plays a crucial role in regulating cancer cell survival, proliferation, invasion, as well as angiogenesis. This pathway is closely regulated by extracellular signals from the cell membrane to the nucleus, governed by a series of phosphorylation events orchestrated by upstream kinases with common somatic point mutations seen in the BRAF (66%) and NRAS (15%) genes (18). In fact, nearly 80%-90% of the BRAF mutations display one specific amino acid substitution, valine to glutamic acid (BRAFV600E); however, other activation mutations are also reported, including BRAFV600K and BRAFV600R (19, 20). The activated BRAF kinase phosphorylates and activates MEK, which is upstream of extracellular signal-regulated kinases (ERK1/2). ERK1/2 regulates multiple functions, such as gene expression, metabolism, and cytoskeletal functions, and is shown to be activated in numerous metastatic cancers. A specific example is seen in melanoma, in which BRAF mutations further activate the MAPK pathway. Moreover, administration of a MAPK inhibitor in melanoma BM cell lines with a BRAFV600E mutation resulted in inhibition of growth (21).

Recent advancements in immunotherapies targeting immune checkpoints have significantly improved survival of cancer patients. Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1)/PD-L1 represent the main class of immunotherapeutic targets for immune checkpoint inhibitors that provides notable efficacy in many cancers. The immunomodulatory role of MAPK and STAT3 signaling has been increasingly recognized; however, their role in regulation of PD-L1 is now beginning to be understood (22).

This study examined the involvement of STAT3 and MAPK pathways, along with the p53 status, in the immunoresponse of metastatic brain tumors. The assessment included an examination of genomic and protein expression, as well as functional analysis utilizing inhibitors of the STAT3 and MAPK pathways.

Materials and Methods

Tumor samples and cell lines. Twenty-six samples of metastatic brain tumors, with primary sites of origin from the lung, breast, colorectal, and melanoma, were obtained from the Department of Pathology, Westchester Medical Center, Valhalla, NY, USA in accordance with the Institutional Review Board guidelines. Samples of human metastatic breast cancer cell line MDA-MB-231 [American Tissue Culture Collection (ATCC), Manassas, VA, USA] were grown in Roswell Park Memorial Institute (RPMI) media with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/amphotericin and maintained in a humidified incubator at 37°C with 5% CO2. The human glioblastoma cell line U87 (ATCC), grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and 1% penicillin/streptomycin/amphotericin, was used as astrocytic media.

Immunohistochemistry. The routine immunohistochemical (IHC) technique was utilized to determine the expression of mutant p53 (p53mt), phospho-STAT3 (pSTAT3Tyr705), phospho-p44/42 MAPK (pERK1/2Thr202/Tyr204), and PD-L1. Metastatic brain tumor samples were incubated overnight with antibodies directed against pSTAT3Tyr705, pERK1/2Thr202/Tyr204, p53mt, and PD-L1 (Cell Signaling Technology Inc, Beverly, MA, USA). Super Picture Poly-HRP conjugate (Invitrogen) was applied for 15 min, followed by DAB substrate solution (Invitrogen) and a running tap water wash. The secondary antibody alone was used as a negative control. To maximize contrast, the samples were counterstained with hematoxylin (Mayer’s hematoxylin; DakoCytomation, Carpinteria, CA, USA). Three random microscope fields were imaged for each sample, and staining was considered positive if grade three or greater staining was achieved. Each stained section was examined by two investigators.

Gene-expression profiling analysis. Total cellular RNA was extracted from samples of metastatic brain tissue (primary lung adenocarcinoma) using the TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Hybridization of tumor sample RNA against normal lung sample RNA was performed on cDNA microarray glass and scanned with Scan Array Lite (Perkin Elmer, Waltham, MA, USA). Fluorescence intensities were calculated using the Iobion Genetraffic software (Iobion, Informatics, La Jolla, CA, USA). The relative expression level of p53, STAT3, and MAPK-associated genes in metastatic brain tumor tissue against the normal lung tissue was assessed as described by Zohrabian et al. (5).

Protein isolation and western blot analysis. Breast cancer cell line, MDA-MB-231 (ATCC), with tendency for cerebral metastasis, was treated with the STAT3 inhibitor NSC74859 (300 nM; Tocris, Minneapolis, MN, USA) or the MAPK/ERK1/2 inhibitor U0126 (20 nM; Tocris) in both regular as well as astrocytic media at two- and six-hour time points. Vehicle-treated cells were used as a control. Protein was extracted from cells using whole cell lysis buffer [1% Triton X-100, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA containing 1% phosphatase & protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)]. Bradford protein assay (Bio-Rad, Hercules, CA, USA) was utilized to spectroscopically determine the protein concentrations at 595 nm using the Multiscan FC Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples (50 μg) were run onto a 10% SDS-PAGE gel and electrotransferred to a polyvinylidene difluoride (PVDF) membrane. The blot was incubated in a 5% nonfat dry milk-blocking solution (Bio-Rad) and then incubated with primary antibodies targeted against pERK1/2Thr202/Tyr204 (Cell Signaling Technology). Bands were detected by chemiluminescence using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Blots were stripped with a stripping reagent (EMD Chemicals, Billerica, MA, USA) and reincubated with antibodies targeted against total ERK (Cell Signaling Technology). Experiments were repeated three times, and densitometric analysis for phospho- and total-ERK1/2 (tERK) were quantified using ImageJ (NIH, Bethesda, MD, USA). Results were calculated as the ratio of the mean pERK1/2Thr202/Tyr204 quantification to the respective tERK values and presented as mean±SEM relative to corresponding controls.

Cell proliferation assays. Starved metastatic breast cancer cell line, MDA-MB-231, was treated with the STAT3 inhibitor NSC74859 (300 nM; Tocris) or the MAPK/ERK1/2 inhibitor U0126 (20 nM; Tocris) for 24 h in both regular media as well as astrocytic media and assessed using the MTT Cell Growth Assay Kit (Chemicon International Inc., Temecula, CA, USA). A Multiscan FC Microplate Reader (Thermo Fisher Scientific) was utilized to measure absorbance (595 nm and 620 nm). Vehicle treatment was given to controls. Final results are presented as the mean±SEM.

Migration assays. Scratch wound healing assays were used to assess cell migration in the presence of the STAT3 inhibitor NSC74859 (300 nM; Tocris) or the MAPK/ERK1/2 inhibitor U0126 (20 nM; Tocris). Vehicle-treated cells were used as a control. A monolayer of MDA-MB-231 cells was wounded, and cell movement was subsequently monitored over a period of 24 h using the Axiovert 200 microscope (ZEISS, Oberkochen, Germany) at 3.6x magnification. The migration rate was determined by measuring the mean distance between borderlines of the wound. Final results are presented as the mean±SEM.

Statistical analysis. Analysis of variance (ANOVA) tests followed by Student’s t-test (unpaired, 2-tailed) were used to evaluate significant variations between control and experimental results. Significance was indicated by a p-value less than 0.05.

Results

Immunohistochemistry demonstrates mutant p53, phosphorylated STAT3, phosphorylated MAPK (ERK1/2), and PD-L1 in metastatic brain tumors. The expression of p53mt, pSTAT3Tyr705, pERK1/2Thr202/Tyr204, and PD-L1 was assessed in human metastatic brain tumor samples originating from multiple primary sources, including the lung, breast, colorectal, and melanoma. High expression of p53mt, pSTAT3Tyr705, pERK1/2Thr202/Tyr204, and PD-L1 was seen in tumor samples (Figure 1). A detailed analysis revealed that while p53mt and PD-L1 expression was seen throughout the tumor tissue, the expression of pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 was confined largely to the tumor margin, implying the roles of these proteins in establishing and facilitating the process of metastasis. There was significant overlap in expression of p53mt, pSTAT3Tyr705, and pERK1/2Thr202/Tyr204 in the tumor samples. Mutant p53 was expressed in 81% of BMs, while 86% and 63% expressed pERK1/2Thr202/Tyr204 and pSTAT3Tyr705, respectively. There was a 62% overlap between p53mt and pERK1/2Thr202/Tyr204 and a 50% overlap between each pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 with p53mt.

Figure 1.
Figure 1.

Immunohistochemical analysis of the expression of p53mt, pSTAT3Tyr705, pERK1/2Thr202/Tyr204, and PD-L1 in metastatic brain tumors. Metastatic brain tumors demonstrated high and intense levels of p53mt expression uniformly throughout the tumor areas. pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 were intense and exclusively present at the tumor margins. PD-L1 expression was present in the majority of brain tumor samples. Venn diagram analysis of p53mt, pSTAT3Tyr705, and pERK1/2Thr202/Tyr204 represents a significant overlap between the expression of these proteins in brain metastatic tissue (see text for details). p53mt: Mutant p53; PD-L1: programmed death ligand 1; H&E: hematoxylin and eosin.

Gene-expression profiling demonstrates multiple p53-, STAT3, and MAPK-related gene mutations in metastatic brain tumors. Figure 2 presents the up-regulated (left panel) or down-regulated (right panel) genes associated with aberrant p53, STAT3, and MAPK. p53-related genes altered in BM tumors were genes associated with apoptosis (p53-regulated apoptosis-inducing protein 1/p53AIP1 and p53-inducible nuclear protein1/p53DINP1). Furthermore, altered genes associated with p53 were related to G2 arrest stage of the cell cycle. In addition, MDM4 (MDMX), a regulator of p53, was also altered. Gene expression analysis demonstrated alterations in 32 STAT3- and MAPK-associated genes. Gene alterations associated with MAPK were mainly involved in regulating the MAPK pathway, such as MAP3K11, a gene that controls the activity of many kinases, including Rho family GTPase (guanosine triphosphatase) that regulates nuclear factor-Embedded ImageB (NF-Embedded ImageB), an inflammatory marker. Gene alterations associated with STAT3 included up-regulation of stathmin-1 and stathmin-like 3, which control microtubule dynamics in STAT3-related cell migration, and changes in cytokine-related genes, suggesting altered immune responses. The most up-regulated gene associated with STAT3 was synaptotagmin VII (SYT7).

Figure 2.
Figure 2.

Gene expression profiling revealed numerous gene alterations associated with aberrant p53, STAT3, and MAPK (see text for details).

Expression of phosphorylated ERK after administration of STAT3 and MAPK inhibitors. The expression of phospho- and total-ERK (tERK) was assessed in human metastatic breast cancer cells with a tendency to metastasize to the brain, following treatment with STAT3 and MAPK (ERK1/2) inhibitors. Expression of pERK1/2Thr202/Tyr204 relative to tERK was significantly decreased in metastatic cells following treatment with the MAPK inhibitor in both regular and astrocytic media at the two- and six-hour time points (Figure 3A). In regular media, expression of activated ERK was significantly suppressed at 2- and 6-hours following administration of U0126 (both p≤0.01). A similar effect was seen when the cells were grown in astrocytic media (both p≤0.01). In both media, effects were more pronounced at 6 h than at 2 h. Following treatment with the STAT3 inhibitor NSC74859, initially, at 2 h, there was an increase in pERK1/2Thr202/Tyr204 in both regular as well as astrocytic media; however, in astrocytic media, this effect was more pronounced. On the contrary, there was significant suppression of pERK1/2Thr202/Tyr204 expression at 6 h after administration of the STAT3 inhibitor in both regular and astrocytic media (both p≤0.05), though to a lesser degree than in cells treated with MAPK inhibitor.

Figure 3.
Figure 3.

Phosphorylated ERK (pERK1/2Thr202/Tyr204) expression is influenced by the STAT3 and MAPK signaling pathways. A) Relative density of phosphorylated ERK (pERK1/2Thr202/Tyr204) compared to total ERK (tERK) in metastatic breast cancer cells after treatment with STAT3 or ERK1/2 inhibitors. ERK1/2 inhibitor (U0126) showed significant reduction in pERK1/2Thr202/Tyr204 at 2 or 6 h in both regular (Reg) and astrocytic (Astro) media. Despite initial increased expression of pERK1/2Thr202/Tyr204 after STAT3 inhibitor (NSC74859) treatment at 2 h, there was significant suppression of expression at 6 h in both media, however notably less than the U0126 treatment. Significance was determined at *p≤0.05; **p≤0.01. B) Schematic of the STAT3 and MAPK signaling pathways. Activated EGF initiates signal through its EGFR to activate RAS-GTP dimer and recruits BRAF/MEK. Activated MEK1/2 phosphorylates ERK1/2, which generates responses to the growth factor signal and other external factors; it can also stimulate the STAT3 pathway. Activated STAT and ERK can localize to the nucleus (DNA) to regulate transcription of various genes, including the gene encoding PD-L1 protein. MAPK: Mitogen-activated protein kinase; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; GTP: guanosine triphosphate; PD-L1: programmed death ligand 1; JAK: Janus kinase; CD274: cluster of differentiation 274; RNA: ribonucleic acid; TF: transcription factor; P: phosphorylated.

Effect of STAT3 and MAPK inhibitors on proliferation of metastatic cells. Proliferation of brain metastatic breast cancer cells was measured utilizing MTT analysis in regular or astrocytic environments. Results demonstrated augmented growth in astrocytic media, with cell viability increased by 20.4% as compared to that in regular media (p≤0.05) (Figure 4A). Administration of the STAT3 inhibitor, NSC74859 (300 nM), for 24 h significantly decreased cell viability under both conditions by 34.2% in regular media and 50.9% in astrocytic media (both p≤0.01). Administration of the MAPK inhibitor, U0126 (20 nM), also caused a suppression in cell viability by 29.0% and 63.3% (both p≤0.01) in regular and astrocytic media, respectively.

Figure 4.
Figure 4.

Cellular viability and migration of metastatic breast cancer cells after treatment with STAT3 or ERK1/2 inhibitors. A) Proliferation and viability after treatment with ERK1/2 inhibitor (U0126) and STAT3 inhibitor (NSC74859) was significantly decreased in cells grown in regular (left panel) or astrocytic media (right panel). B) Migration was significantly decreased following U0126 administration in cells grown in regular media and stagnated in cells grown astrocytic media. NSC74859 was less effective at reducing migration. C) Scratch wound images at 0 and 24 h after STAT3 and ERK1/2 inhibitors compared to control in cells grown in regular or astrocytic media. Significance was determined at *p≤0.05; ***p<0.01; $trend towards significance.

Effect of STAT3 and MAPK inhibitors on migration of metastatic cells. Motility of breast cancer cells (MDA-MB-231), with tendency for cerebral metastases, was measured over a period of 24 h in regular or astrocytic media using scratch wound migration assay (Figure 4C) following treatment with the STAT3 inhibitor NSC74859 (300 nM), or the MAPK inhibitor U0126 (20 nM). Treatment with NSC74859 caused a trend towards suppression of migration in astrocytic media (p=0.07), but migration remained unaltered in regular media (p=0.19) (Figure 4B). MAPK inhibitor (U0126) treatment in regular media caused a trend towards reduced migration compared to controls (p=0.07); however, in astrocytic media, U0126 treatment caused a significant reduction in migration. In fact, a complete stagnation in migration was seen in U0126-treated cells as compared to controls in astrocytic media (p≤0.01).

Discussion

The results of this study demonstrated the significant presence of mutant p53, activated STAT3, activated ERK1/2, and PD-L1 in metastatic brain tumor samples. Expression of mutant p53 was seen throughout the tumor area; similarly, PD-L1 expression was also seen all over the tumor area. It was evident that pSTAT3Tyr705 as well as pERK1/2Thr202/Tyr204 were expressed heavily at the margin of tumors, implicating their potential roles in tumor growth, metastasis, and immune response.

The gene expression profile of BM tumors originating from adenocarcinoma of the lung revealed the expression of genes under different pathways and functions, possibly involving known as well as several unknown signaling pathways. Additionally, several known genes relating to tumor metastasis to the brain were also noted. As shown in Figure 1, our study demonstrated that mutant p53 is present in a significant proportion of brain metastases, lending further evidence to the role of mutant p53 in brain metastases (5, 23). p53 is a transcription factor that plays a critical role in regulating cell growth, DNA repair, and apoptosis in response to stressful conditions. Expression of mutant p53 was widespread in the tumor, and the intensity of expression was noticeably high (Figure 1). These results are in accordance with studies demonstrating high occurrence of TP53 mutation in brain metastatic lesions, showing presence of complex mutations, including nonsense, deletions, insertions, etc. (13). The most common p53 gene missense mutations in human cancer are found in exons 5-9 and are often termed ‘hotspot mutations’ (12). TP53 mutations found in cancers encode mutant p53 proteins displaying a gain of function, endowing them with oncogenic properties capable of morphologically transforming primary cells to a malignant phenotype. p53 gene alteration is achieved by various means and appears to be a common event in malignant disease, though the incidence of p53 gene mutation differs noticeably between tumor types (11, 13). For CNS metastases, the most common primary malignancies originate from the lung and breast (24). Generally, both triple-negative breast cancer, as well as hormone receptor/human epidermal growth factor receptor 2 (HER2)-positive brain metastases, appear to possess a higher number of p53 mutations (13). In addition, studies demonstrated a significant trend for mutual exclusivity between truncated mutations in TP53 in adenocarcinoma of the lung (25). A study comparing the p53 mutations in CNS metastatic lesions with matched primary tumors revealed the presence of clonal selection as well as development of novel p53 mutations (missense and complex) in the metastatic lesions, suggesting that the progression from a primary breast carcinoma to brain metastasis may consist of both “driver” as well as evolved p53 mutations (14). Our results showed alterations in p53-associated genes, including increased gene expression of p53, up-regulation of Reprimo, a candidate gene mediating p53-dependent G2 arrest through involvement of cyclin B1 and the Cdc2 pathway, and down-regulation of p53AIP1, a novel p53 target that mediates apoptosis (Figure 2). In addition, there was decreased expression of tumor protein p53DINP1, which plays a role in p53-dependent apoptosis, and down-regulation of MDM4 (MDMX) expression, a noted negative regulator of p53.

Our results demonstrated high expression of pSTAT3Tyr705 in the tumor margin area. While the role of STAT3 in brain metastasis is not well established, the presence of STAT3 in reactive astrocytes surrounding brain metastatic cells has been shown to contribute to the pro-metastatic nature of the lesion (26). This suggests that BM cells benefit from reactive astrocytes to modulate an effective immune response. In fact, presence of activated STAT3 in reactive astrocytes of patients with BM correlates with reduced survival because of intracranial metastases (26). Furthermore, presence of activated STAT3 is shown in astrocytes and microglia in areas of gliosis as well as areas of radiation-induced necrosis in BM specimens (27). Consequently, inhibiting STAT3 signaling could reduce brain metastasis, even in advanced stages (28). One of the genes highly up-regulated by STAT3 in our study is synaptotagmin VII (SYT7) (Figure 2). SYT7 appears to play a significant role in EMT and is associated with metastasis in many cancers (29). The process of BM occurring via EMT has previously been described in literature (6).

Functional analysis was performed to evaluate the utility of targeting STAT3 and MAPK in BM tumor cells. Assessment of cell proliferation and migration revealed that cells grown in astrocytic media were more viable and highly migratory as compared to cells in regular media. The inhibition of the STAT3 or MAPK pathway suppressed cell proliferation under both growing conditions, albeit greater in astrocytic media. Migration was suppressed with administration of the STAT3 inhibitor and completely stagnated by the MAPK (ERK1/2) inhibitor.

STAT3 is known to be activated by numerous cytokines, growth factors, and oncogenic proteins, including epidermal growth factor, platelet-derived growth factor, vascular endothelial growth factor, basic fibroblast growth factor, interleukin (IL)-6, proto-oncogene Src, and Ras, suggesting that STAT3 signaling may be one of the common pathways involved in regulating cancer metastasis. Several lines of evidence indicate that constitutively activated STAT3 influences metastasis. Activation of STAT3 in thymic epithelial tumors, colorectal adenocarcinoma, and cutaneous squamous cell carcinoma correlates with lymph node metastasis (30, 31). Furthermore, activation of STAT3 in renal cell carcinoma is associated with distant metastatic disease (32). In addition, STAT3 activity is up-regulated in human melanoma specimens, and higher levels of activated STAT3 protein have been detected in melanoma brain metastases when compared to a primary cohort (5). Also, using in vivo model systems of liver, lung, and brain metastases, the roles and mechanisms of activated STAT3 in metastases have been examined. Studies have shown that STAT3 represses the p53 gene transcription rate by binding to the p53 promoter and subsequently affecting p53-mediated tumor cell apoptosis. Moreover, STAT3 may contribute to tumor cell migration and invasion through transcription-independent pathways as the STAT3 protein is localized to focal adhesions with focal adhesion kinases (FAK) and paxillin to modulate the invasiveness of ovarian cancer cells (31). Importantly, STAT3 signaling plays a pivotal role in the regulation of tumor immunity, as STAT3 is constitutively activated in both tumor cells and tumor-associated immune cells (33, 34). Moreover, STAT3 signaling inhibits the expression of inflammatory cytokines and chemokines [interferon (IFN)-β, tumor necrosis factor-α, IL-6, and IFN-γ-inducible protein-10] that activate antitumor innate and adaptive immunity and dendritic cells, leading to tumor-specific T-cell responses (35). STAT3 may interact with various molecules, including MAPK in the inflammatory response.

Tumor cell proliferation and survival involve down-regulation of wild-type p53 (WTp53) expression as well as increase in STAT3 activity. Conversely, WTp53 reduces STAT3 phosphorylation and DNA-binding activity in breast and prostate cancer cells, and pSTAT3Tyr705 activity suppresses TP53 expression, illustrating their negative regulation of one another (36, 37). This reciprocal regulation can be explained by the opposing biological roles of both factors, since pSTAT3Tyr705 functions as an oncogene (38). Mutations of p53 are reported to occur early and are involved in the tumor initiation process; however, p53 mutations in cancers can also develop late in the tumorigenic process, contributing to tumor progression and metastasis (39). Moreover, the loss of WTp53 function as well as accumulation of p53mt may contribute to the maintenance of STAT3-mediated tumor cell survival and expansion (40). Our results show that a significant number of BM tumors (about 50%) display both p53mt and pSTAT3Tyr705 (Figure 1), suggesting a potential role for p53mt and pSTAT3Tyr705 in the initiation and maintenance of brain metastases.

MAPK, specifically ERK1/2, signaling pathways are extensively activated in various cancers, including breast cancer. Numerous studies have demonstrated that activation of ERK1/2 promotes cell survival and growth; alternatively, abnormal ERK1/2 activation can augment apoptotic processes (41, 42). The genetically altered upstream pathway of ERK1/2, including activated Ras and MEK1/2, is critical to the BM and EMT process. The MAPK pathway plays a crucial role in regulating cancer cell survival, proliferation, invasion, as well as angiogenesis, and it is regulated by extracellular signals, such as growth factors, etc., controlled by a series of phosphorylation events orchestrated by upstream kinases as shown by somatic point mutations in BRAF (66%) and NRAS (15%) genes (18). The activated BRAF kinase phosphorylates and activates MEK1/2, an upstream kinase of ERK1/2, which regulates multiple physiological functions, including metabolism, cell proliferation, and cytoskeletal dynamics; ERK1/2 is shown to be activated in numerous primary as well as metastatic cancers, suggesting its role in initiation and progression of cancer. p53 status in cells can affect the activation of ERK1/2 in response to external factors; our earlier study showed that 12-O-tetradecanoylphorbol-13-acetate (TPA)-modulated Ras/ERK signaling pathways work differently depending on the p53 status of the cells (43). Mutant p53 shows only a transient ERK activation in response to tumorigenic TPA, while WTp53 cells had a sustained activation of ERK (43). In addition, it has been shown that suppression of the mTOR pathway can activate ERK1/2 via the MEK1/2 pathway, involving Ras/ERK (44). These observations suggest the tumor cells containing p53 mutation have discrete responses to the inhibition of interdependent pathways. Western blot analysis of ERK1/2 expression revealed that treatment with a MAPK inhibitor (U0126), as well as a STAT3 inhibitor (NSC74859), caused a significant reduction in levels of pERK1/2Thr202/Tyr204 (Figure 3A). Therefore, inhibition of STAT3 can also inhibit MAPK, similar to ERK1/2 inhibitor (U0126), demonstrating a crosstalk between STAT3 and MAPK. In fact, STAT and MAPK can interact in regulation of PD-L1 status (45).

Activated MAPK can influence STAT3 signaling in regulation of PD-L1 (Figure 3B). Stimulation with insulin-like growth factor (IGF), or IFN-γ, increases PD-L1 expression in lung adenocarcinoma; however, inhibition of the ERK1/2 pathway prevents this induction of PD-L1 expression (45). Studies have also provided evidence that KRAS-induced MAPK signaling can lead to activation of STAT3 signaling and localization to the nucleus, in turn causing production of PD-L1 protein (45). Our study provides a foundation for understanding the interdependence of the ERK1/2 and STAT3 pathways in BM signaling; however, it remains to be seen whether this interaction occurs during the initiation of BM.

Conclusion

This study demonstrates the expression of activated STAT3 and MAPK at the margins of BM tumors, suggesting their involvement not only in proliferation but also in immunoresponse. These findings, therefore, provide experimental evidence to suggest that targeting the STAT3/MAPK pathways may be an effective treatment strategy to induce immuno-responsiveness for the treatment of brain metastasis.

Footnotes

  • Authors’ Contributions

    SZ contributed to data curation, formal analysis, investigation, writing, reviewing & editing. ES contributed to investigation, writing, reviewing & editing. AD contributed in experimental process and data acquisition, JW, SH, and CG contributed to funding acquisition, supervision, reviewing & editing. MJ-U contributed to funding acquisition, conceptualization, data curation, formal analysis, funding acquisition, project administration, supervision, writing, reviewing & editing.

  • Conflicts of Interest

    All Authors (SZ, ES, AD, JW, SH, CG, MJU) declare no conflicts of interest in relation to this study.

  • Funding

    This research was supported by funds from the Advanced Research Foundation and funds from The Rockefeller Foundation.

  • Received November 30, 2023.
  • Revision received December 13, 2023.
  • Accepted December 15, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Brain Metastases Are Regulated by Immuno-inflammatory Signaling Pathways Governed by STAT3, MAPK and Tumor Suppressor p53 Status: Possible Therapeutic Targets
SABRINA L. ZELLER, ERIS SPIROLLARI, ALIS J. DICPINIGAITIS, JOHN V. WAINWRIGHT, SIMON J. HANFT, CHIRAG D. GANDHI, MEENA JHANWAR-UNIYAL
Anticancer Research Jan 2024, 44 (1) 13-22; DOI: 10.21873/anticanres.16783
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