KLF4 overcomes tamoxifen resistance by suppressing MAPK signaling pathway and predicts good prognosis in breast cancer

Tamoxifen resistance represents a daunting challenge to the successful treatment for breast cancer. Krüppel-like factor 4 has critical roles in the development and progression of breast cancer, but its expression, function and regulation in the efficacy of TAM therapy in breast cancer have yet to be investigated. Here, we examined the clinical significance and biologic effects of KLF4 in breast cancer. Firstly, higher expression of KLF4 correlated with increased TAM sensitivity in breast cancer cells, and analysis of GEO datasets indicated that KLF4 expression was positively correlated with ERα and enhanced expression of KLF4 sensitized breast cancer patients to endocrine therapy. Knockdown of KLF4 in MCF-7 and BCAP37 cells led to increased TAM resistance, while ectopic KLF4 expression promoted the responsiveness to TAM in T47D and TAM-resistant MCF-7/TAM cells. Secondly, ectopic KLF4 overexpression suppressed MCF-7/TAM cell growth, invasion and migration. Moreover, KLF4 expression was down-regulated in breast cancer tumor tissues and high expression of KLF4 was associated with favorable outcomes. Mechanistically, KLF4 may enhance the responsiveness of breast cancer cells to TAM through suppressing mitogen-activated protein kinase (MAPK) signaling pathway. We found that ERK and p38 were more activated in MCF-7/TAM compared with MCF-7, and treatment with MAPK-specific inhibitors significantly suppressed cell viability. Knockdown of KLF4 activated ERK and p38 and drove MCF-7 cells to become resistant to TAM. Conversely, overexpression of KLF4 in MCF-7/TAM cells suppressed ERK and p38 signaling and resulted in increased sensitivity to TAM. Therefore, our findings suggested that KLF4 contributed to TAM sensitivity in breast cancer via phosphorylation modification of ERK and p38 signaling. Collectively, this study highlighted the significance of KLF4/MAPK signal interaction in regulating TAM resistance of breast cancer, and suggested that targeting KLF4/MAPK signaling may be a potential therapeutic strategy for breast cancer treatment, especially for the TAM-resistant patients.     

AhR ligand aminoflavone suppresses α6-integrin–Src–Akt signaling to attenuate tamoxifen resistance in breast cancer cells

More than 40% of patients with luminal breast cancer treated with endocrine therapy agent tamoxifen demonstrate resistance. Emerging evidence suggests tumor initiating cells (TICs) and aberrant activation of Src and Akt signaling drive tamoxifen resistance and relapse. We previously demonstrated that aryl hydrocarbon receptor ligand aminoflavone (AF) inhibits the expression of TIC gene α6-integrin and disrupts mammospheres derived from tamoxifen-sensitive breast cancer cells. In the current study, we hypothesize that tamoxifen-resistant (TamR) cells exhibit higher levels of α6-integrin than tamoxifen-sensitive cells and that AF inhibits the growth of TamR cells by suppressing α6-integrin–Src–Akt signaling. In support of our hypothesis, TamR cells and associated mammospheres were found to exhibit elevated α6-integrin expression compared with their tamoxifen-sensitive counterparts. Furthermore, tumor sections from patients who relapsed on tamoxifen showed enhanced α6-integrin expression. Gene expression profiling from the TCGA database further revealed that basal-like breast cancer samples, known to be largely unresponsive to tamoxifen, demonstrated higher α6-integrin levels than luminal breast cancer samples. Importantly, AF reduced TamR cell viability and disrupted TamR mammospheres while concomitantly reducing α6-integrin messenger RNA and protein levels. In addition, AF and small interfering RNA against α6-integrin blocked tamoxifen-stimulated proliferation of TamR MCF-7 cells and further sensitized these cells to tamoxifen. Moreover, AF reduced Src and Akt signaling activation in TamR MCF-7 cells. Our findings suggest elevated α6-integrin expression is associated with tamoxifen resistance and AF suppresses α6-integrin–Src–Akt signaling activation to confer activity against TamR breast cancer.     

Src Drives Growth of Antiestrogen Resistant Breast Cancer Cell Lines and Is a Marker for Reduced Benefit of Tamoxifen Treatment

The underlying mechanisms leading to antiestrogen resistance in estrogen-receptor α (ER)-positive breast cancer is still poorly understood. The aim of this study was therefore to identify biomarkers and novel treatments for antiestrogen resistant breast cancer. We performed a kinase inhibitor screen on antiestrogen responsive T47D breast cancer cells and T47D-derived tamoxifen and fulvestrant resistant cell lines. We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells. Furthermore western blot analysis showed increased expression and phosphorylation of Src in the resistant cells and that dasatinib inhibited phosphorylation of Src and also signaling via Akt and Erk in all cell lines. Immunoprecipitation revealed Src: ER complexes only in the parental T47D cells. In fulvestrant resistant cells, Src formed complexes with the Human Epidermal growth factor Receptor (HER)1 and HER2. Neither HER receptors nor ER were co-precipitated with Src in the tamoxifen resistant cell lines. Compared to treatment with dasatinib alone, combined treatment with dasatinib and fulvestrant had a stronger inhibitory effect on tamoxifen resistant cell growth, whereas dasatinib in combination with tamoxifen had no additive inhibitory effect on fulvestrant resistant growth. When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival. In conclusion, Src was identified as target for treatment of antiestrogen resistant T47D breast cancer cells. For tamoxifen resistant T47D cells, combined treatment with dasatinib and fulvestrant was superior to treatment with dasatinib alone. Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen.     

Downregulation of CXXC Finger Protein 4 Leads to a Tamoxifen-resistant Phenotype in Breast Cancer Cells Through Activation of the Wnt/β-catenin Pathway

Tamoxifen is a successful endocrine therapy drug for estrogen receptor–positive (ER+) breast cancer. However, resistance to tamoxifen compromises the efficacy of endocrine treatment. In the present study, we identified potential tamoxifen resistance–related gene markers and investigated their mechanistic details. First, we established two ER + breast cancer cell lines resistant to tamoxifen, named MCF-7/TMR and BT474/TMR. Gene expression profiling showed that CXXC finger protein 4 (CXXC4) expression is lower in MCF-7/TMR cells than in MCF-7 cells. Furthermore, CXXC4 mRNA and protein expression are lower in the resistant cell lines than in the corresponding parental cell lines. We also investigated the correlation between CXXC4 and endocrine resistance in ER + breast cancer cells. CXXC4 knockdown accelerates cell proliferation in vitro and in vivo and renders breast cancer cells insensitive to tamoxifen, whereas CXXC4 overexpression inhibits cancer cell growth and increases tamoxifen sensitivity of resistant cells. In addition, we demonstrated that CXXC4 inhibits Wnt/β-catenin signaling in cancer cells by modulating the phosphorylation of GSK-3β, influencing the integrity of the β-catenin degradation complex. Silencing the CXXC4 gene upregulates expression of cyclinD1 and c-myc (the downstream targets of Wnt signaling) and promotes cell cycle progression. Conversely, ectopic expression of CXXC4 downregulates the expression of these proteins and arrests the cell cycle in the G0/G1 phase. Finally, the small-molecule inhibitor XAV939 suppresses Wnt signaling and sensitizes resistant cells to tamoxifen. These results indicate that components of Wnt pathway that are early in response to tamoxifen could be involved as an intrinsic factor of the transition to endocrine resistance, and inhibition of Wnt signaling may be an effective therapeutic strategy to overcome tamoxifen resistance.     

Kinase-specific phosphorylation of the estrogen receptor changes receptor interactions with ligand, deoxyribonucleic acid, and coregulators associated with alterations in estrogen and tamoxifen activity

Posttranslational modifications of the estrogen receptor (ER) are emerging as important regulatory elements of cross talk between different signaling pathways. ER phosphorylation, in particular, has been implicated in the ligand-independent effects of ER and in tamoxifen resistance of breast tumors. In our studies, Western immunoblot analysis of endogenous ER in parental MCF-7 cells reveals specific, ligand-dependent phosphorylations at S118 and S167, with this ligand dependence being lost in tamoxifen-resistant, MCF-7 Her2/neu cells. Using highly purified components and sensitive fluorescence methods in an in vitro system, we show that phosphorylation by different kinases alters ER action through distinct mechanisms. Phosphorylation by Src and protein kinase A increases affinity for estradiol (E2), whereas ER phosphorylation by MAPK decreases trans-hydroxytamoxifen (TOT) binding. Affinity of ER for the consensus estrogen response element is also altered by phosphorylation in a ligand-specific manner, with decrease in affinity of MAPK- and Src-phosphorylated ER in the presence of TOT. ER phosphorylation by MAPK, AKT, or protein kinase A increases recruitment of steroid receptor coactivator 3 receptor interaction domain to the DNA-bound receptor in the presence of E2. Taken together, these results suggest that ER phosphorylation alters receptor functions (ligand, DNA, and coactivator binding), effecting changes that could lead to an increase in E2 agonism and a decrease in TOT antagonistic activity, reflecting changes encountered in tamoxifen resistance in endocrine therapy of breast cancer.     

Activation function-1 domain of estrogen receptor regulates the agonistic and antagonistic actions of tamoxifen

The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ERalpha-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ERalpha, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ERalpha, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ERalpha regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.     

Elevated expression of CUEDC2 protein confers endocrine resistance in breast cancer

Endocrine resistance is a major obstacle to hormonal therapy for breast cancers. Although reduced expression of estrogen receptor-α (ER-α) is a known contributing factor to endocrine resistance, the mechanism of ER-α downregulation in endocrine resistance is still not fully understood. Here we report that CUE domain-containing protein-2 (CUEDC2), a ubiquitin-binding motif-containing protein, is a key factor in endocrine resistance in breast cancer. We show that CUEDC2 modulates ER-α protein stability through the ubiquitin-proteasome pathway. Through the study of specimens from a large cohort of subjects with breast cancer, we found a strong inverse correlation between CUEDC2 and ER-α protein expression. Notably, subjects with tumors that highly expressed CUEDC2 had poor responsiveness to tamoxifen treatment and high potential for relapse. We further show that ectopic CUEDC2 expression impaired the responsiveness of breast cancer cells to tamoxifen. Therefore, our findings suggest that CUEDC2 is a crucial determinant of resistance to endocrine therapies in breast cancer.     

Characterization of BCAR4, a novel oncogene causing endocrine resistance in human breast cancer cells

Resistance to the antiestrogen tamoxifen remains a major problem in the management of estrogen receptor-positive breast cancer. Knowledge on the resistance mechanisms is needed to develop more effective therapies. Breast cancer antiestrogen resistance 4 (BCAR4) was identified in a functional screen for genes involved in tamoxifen resistance. BCAR4 is expressed in 27% of primary breast tumors. In patients treated with tamoxifen for metastized disease high BCAR4 mRNA levels are associated with reduced clinical benefit and progression-free survival. Regarding tumor aggressiveness high BCAR4 mRNA levels are associated with a shorter metastasis free survival and overall survival. In the present study, we investigated the role of BCAR4 in endocrine resistance. Forced expression of BCAR4 in human ZR-75-1 and MCF7 breast cancer cells resulted in cell proliferation in the absence of estrogen and in the presence of various antiestrogens. Inhibition of estrogen receptor 1 (ESR1) expression with small interfering RNA (siRNA), implied that the BCAR4-induced mechanism of resistance is independent of ESR1. Highly conserved BCAR4 homologues of rhesus monkey, green monkey, and the less conserved common marmoset gene induced tamoxifen-resistant cell proliferation, in contrast to the distant BCAR4 homologues of bovine and rabbit. Injection of BCAR4-expressing ZR-75-1 cells into nude mice resulted in rapidly growing tumors. In silico analysis showed that BCAR4 mRNA is highly expressed in human placenta and oocyte, and absent in other normal tissues. In conclusion, BCAR4 is a strong transforming gene causing estrogen-independent growth and antiestrogen resistance, and induces tumor formation in vivo. Due to its restricted expression, BCAR4 may be a good target for treating antiestrogen-resistant breast cancer.     

Regulation of P53 signaling in breast cancer by the E3 ubiquitin ligase RNF187

The tumor suppressor P53 plays critical role in preventing cancer. P53 is rarely mutated and remains functional in luminal-type breast cancer(1). According to current knowledge, wild-type P53 function is tightly controlled by posttranslational modifications, such as ubiquitination. Several ubiquitin ligases have been shown to regulate P53 ubiquitination and protein stability. Here, we report that RNF187, a RING family ubiquitin ligase, facilitates breast cancer growth and inhibits apoptosis by modulating P53 signaling. RNF187 expression was elevated in breast cancer and correlated with breast cancer survival only in the P53 wild-type groups. Bioinformatic analysis showed that the expression of RNF187 was negatively correlated with the expression of P53 target genes, such as IGFBP3 and FAS, in breast cancer. RNF187 depletion inhibited breast cancer growth and facilitated cell death. RNA sequencing analysis indicated that RNF187 could be an important modulator of P53 signaling. Further experiments showed that RNF187 interacts with P53 and promotes its degradation by facilitating its polyubiquitination in breast cancer cells. Interestingly, the in vitro ubiquitin assay showed that RNF187 can directly ubiquitinate P53 in a manner independent of MDM2. These findings reveal a novel direct P53 regulator and a potential therapeutic target for breast cancer.Subject terms: Breast cancer, Ubiquitylation     

MGMT Inhibition Restores ERα Functional Sensitivity to Antiestrogen Therapy

Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O(6) methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O(6)-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21(cip1/waf1). We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21(cip1/waf1) and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance.     

MTA1 is a novel regulator of autophagy that induces tamoxifen resistance in breast cancer cells

Tamoxifen is commonly used to treat patients with ESR/ER-positive breast cancer, but its therapeutic benefit is limited by the development of resistance. Recently, alterations in macroautophagy/autophagy function were demonstrated to be a potential mechanism for tamoxifen resistance. Although MTA1 (metastasis-associated 1) has been implicated in breast tumorigenesis and metastasis, its role in endocrine resistance has not been studied. Here, we report that the level of MTA1 expression was upregulated in the tamoxifen resistant breast cancer cell lines MCF7/TAMR and T47D/TR, and knockdown of MTA1 sensitized the cells to 4-hydroxytamoxifen (4OHT). Moreover, knockdown of MTA1 significantly decreased the enhanced autophagy flux in the tamoxifen resistant cell lines. To confirm the role of MTA1 in the development of tamoxifen resistance, we established a cell line, MCF7/MTA1, which stably expressed MTA1. Compared with parental MCF7, MCF7/MTA1 cells were more resistant to 4OHT-induced growth inhibition in vitro and in vivo, and showed increased autophagy flux and higher numbers of autophagosomes. Knockdown of ATG7 or cotreatment with hydroxychloroquine, an autophagy inhibitor, restored sensitivity to 4OHT in both the MCF7/MTA1 and tamoxifen resistant cells. In addition, AMP-activated protein kinase (AMPK) was activated, probably because of an increased AMP:ATP ratio and decreased expression of mitochondrial electron transport complex components. Finally, publicly available breast cancer patient datasets indicate that MTA1 levels correlate with poor prognosis and development of recurrence in patients with breast cancer treated with tamoxifen. Overall, our findings demonstrated that MTA1 induces AMPK activation and subsequent autophagy that could contribute to tamoxifen resistance in breast cancer.     

Functional screen for genes responsible for tamoxifen resistance in human breast cancer cells

Antiestrogens, such as tamoxifen, are widely used for endocrine treatment of estrogen receptor-positive breast cancer. However, as breast cancer progresses, development of tamoxifen resistance is inevitable. The mechanisms underlying this resistance are not well understood. To identify genes involved in tamoxifen resistance, we have developed a rapid screening method. To alter the tamoxifen-sensitive phenotype of human ZR-75-1 breast cancer cells into a tamoxifen-resistant phenotype, the cells were infected with retroviral cDNA libraries derived from human placenta, human brain, and mouse embryo. Subsequently, the cells were selected for proliferation in the presence of 4-hydroxy-tamoxifen (OH-TAM) and integrated cDNAs were identified by sequence similarity searches. From 155 OH-TAM-resistant cell colonies, a total of 25 candidate genes were isolated. Seven of these genes were identified in multiple cell colonies and thus cause antiestrogen resistance. The epidermal growth factor receptor, platelet-derived growth factor receptor-alpha, platelet-derived growth factor receptor-beta, colony-stimulating factor 1 receptor, neuregulin1, and fibroblast growth factor 17 that we have identified have been described as key regulators in the mitogen-activated protein kinase pathway. Therefore, this pathway could be a valuable target in the treatment of patients with breast cancer resistant to endocrine treatment. In addition, the putative gene LOC400500, predicted by in silico analysis, was identified. We showed that ectopic expression of this gene, designated as breast cancer antiestrogen resistance 4 (BCAR4), caused OH-TAM resistance and anchorage-independent cell growth in ZR-75-1 cells and that the intact open reading frame was required for its function. We conclude that retroviral transfer of cDNA libraries into human breast cancer cells is an efficient method for identifying genes involved in tamoxifen resistance.     

Identification and validation of SRC and phospho-SRC family proteins in circulating mononuclear cells as novel biomarkers for pancreatic cancer

There is an urgent need to develop novel markers of pancreatic cancer to facilitate early diagnosis. Pancreatic carcinoma is characterized by marked stroma formation with a high number of infiltrating tumor-associated macrophages (TAMs) that originate from circulating mononuclear cells (MNCs). We hypothesized that differential analysis of protein expression and phosphorylation in circulating MNCs from healthy nude mice and nude mice bearing orthotopic human pancreatic cancer would identify a surrogate marker of pancreatic cancer. These differences were analyzed by two-dimensional gel electrophoresis followed by Western blot analysis using antibody against phosphorylated tyrosine proteins (pY). Protein and phosphorylated protein spots of interest were identified by mass spectrometry and validated by Western blot analysis as candidate markers for pancreatic cancer. We found that the expression and phosphorylation of Src family proteins were significantly higher in circulating MNCs from mice bearing pancreatic cancer than in circulating MNCs from healthy mice. TAMs in mice with pancreatic tumors also had higher Src family protein expression and phosphorylation than resident macrophages in the pancreas of healthy mice. The expression and phosphorylation of Src family proteins were correlated with tumor weight; however, increased Src expression and phosphorylation also occurred in MNCs from mice with chronic pancreatitis. This is the first report to explore novel pancreatic tumor markers in circulating MNCs. Although the specificity of the marker for pancreatic cancer was low, it could be used to monitor the disease or to select high-risk patients with chronic pancreatitis.     

Anandamide inhibits adhesion and migration of breast cancer cells

The endocannabinoid system regulates cell proliferation in human breast cancer cells. We reasoned that stimulation of cannabinoid CB1 receptors could induce a non-invasive phenotype in breast metastatic cells. In a model of metastatic spreading in vivo, the metabolically stable anandamide analogue, 2-methyl-2'-F-anandamide (Met-F-AEA), significantly reduced the number and dimension of metastatic nodes, this effect being antagonized by the selective CB1 antagonist SR141716A. In MDA-MB-231 cells, a highly invasive human breast cancer cell line, and in TSA-E1 cells, a murine breast cancer cell line, Met-F-AEA inhibited adhesion and migration on type IV collagen in vitro without modifying integrin expression: both these effects were antagonized by SR141716A. In order to understand the molecular mechanism involved in these processes, we analyzed the phosphorylation of FAK and Src, two tyrosine kinases involved in migration and adhesion. In Met-F-AEA-treated cells, we observed a decreased tyrosine phosphorylation of both FAK and Src, this effect being attenuated by SR141716A. We propose that CB1 receptor agonists inhibit tumor cell invasion and metastasis by modulating FAK phosphorylation, and that CB1 receptor activation might represent a novel therapeutic strategy to slow down the growth of breast carcinoma and to inhibit its metastatic diffusion in vivo.