Regulation of KLF4 by posttranslational modification circuitry in endocrine resistance

Abstract

KLF4 plays an important role in orchestrating a variety of cellular events, including cell-fate decision, genome stability and apoptosis. Its deregulation is correlated with human diseases such as breast cancer and gastrointestinal cancer. Results from recent biochemical studies have revealed that KLF4 is tightly regulated by posttranslational modifications. Here we report a new finding that KLF4 orchestrates estrogen receptor signaling and facilitates endocrine resistance. We also uncovered the underlying mechanism that alteration of KLF4 by posttranslational modifications such as phosphorylation and ubiquitylation changes tumor cell response to endocrine therapy drugs. IHC analyses using based on human breast cancer specimens showed the accumulation of KLF4 protein in ER-positive breast cancer tissues. Elevated KLF4 expression significantly correlated with prognosis and endocrine resistance. Our drug screening for suppressing KLF4 protein expression led to identification of Src kinase to be a critical player in modulating KLF4-mediated tamoxifen resistance. Depletion of VHL (von Hippel-Lindau tumor suppressor), a ubiquitin E3 ligase for KLF4, reduces tumor cell sensitivity to tamoxifen. We demonstrated phosphorylation of VHL by Src enhances proteolysis of VHL that in turn leads to upregulation of KLF4 and increases endocrine resistance. Suppression of Src-VHL-KLF4 cascade by Src inhibitor or enhancement of VHL-KLF4 ubiquitination by TAT-KLF4 (371-420AAa) peptides re-sensitizes tamoxifen-resistant breast cancer cells to tamoxifen treatment. Taken together, our findings demonstrate a novel role for KLF4 in modulating endocrine resistance via the Src-VHL-KLF4 axis.

1. Introduction

Breast cancer is a heterogeneous disease and approximately 70% of human breast cancer patients diagnosed to be estrogen receptor (ER) alpha-positive and hormone-dependent [1]. While endocrine therapy has benefited ER-positive patient survival from breast cancer, a critical challenge of endocrine therapy is the development of drug resistance and recurrence. Tremendous endeavors have been made to determine the molecular mechanisms underlying endocrine resistance [2]. Multiple mechanisms responsible for endocrine resistance have been hypothesized, including deregulation estrogen receptor pathway, cell cycle, cell survival pathway as well as the activation of escape pathways that provide tumors with alternative cell proliferative and survival stimuli [3].

Kruppel-like factor 4 (KLF4), a zinc finger-containing transcription factor, plays important roles in diverse cellular processes, including cell cycle progression, apoptosis, and metabolism as well as stem cell self-renewal and maintenance of pluripotency. KLF4 is one of the core members of the pluripotency transcriptional network, which switches somatic cell into stem cell [4]. Recent studies of carcinogenesis have revealed its dual role as a tumor suppressor or an oncogene, depending on the tissue types and physiological context, with the underlying mechanism of its functional switch remaining largely unknown [5]. While KLF4 is thought to be a tumor suppressor in lung, gastric, colorectal, urothelial, cervical, bladder, prostate and stomach cancers, its oncogenic role has been reported in breast, oral and skin squamous cell carcinomas [6, 7]. Recent studies of the TCGA project further suggested the impact of KLF4 in breast tumor oncogenesis [8-10]. Analyses based on Oncomine database showed a correlation of low KLF4 mRNA levels in breast tumor tissues. However, at protein levels, nuclear accumulation of KLF4 had been reported to be associated with increased risk of recurrence or death in primary breast cancer [6, 7, 11-14]. Furthermore, it was shown that elevated KLF4 expression promotes cell migration and invasion during mammary tumorigenesis [6, 7, 11]. It has reported that KLF4 is regulated by multiple posttranslational modifications such as acetylation, phosphorylation, methylation, SUMOylating, and ubiquitin-depended proteasomal degradation in response to various environmental factors such as estrogen signals and DNA damage [12, 13, 15]. On the other hand, estrogen signal downregulates VHL that in turn causes accumulation of KLF4, which promotes estrogen-induced transactivation, mitogenic effect and cell growth as well as tumorigenesis [12].

Steroids such as 17β-estradiol (E2), via binding to cytoplasmic or membrane-associated estrogen receptors, can rapidly activate intracellular signaling cascades such as c-Src, ERK, PI3K, and STATs. These E2 stimulated phosphorylation alters estrogen signaling and further modulates endocrine resistance [16]. The oncogene Src kinase is a critical component involved in multiple signaling pathways that regulate proliferation, survival, angiogenesis and metastasis [17]. In breast cancer, Src kinase activity plays a critical role in cancer development and further modulates cell survival for endocrine resistant breast cancer cells [18-20]. Moreover, Src kinase regulates both growth factor and E2-stimulated cell growth, and other growth factor signaling such as EGF or IGF-1 [16]. Increased Src activity was showed to enhance breast cancer migration and invasion after long-term treatment with tamoxifen, while blockade of its activity could reverse tamoxifen resistance [19, 21]. Furthermore, Src kinase directly binds and phosphorylates ERα, resulting in promotion of ERα hormone-binding, dimerization, activation as well as drop of affinity between ER and tamoxifen [22, 23]. Our recent studies have revealed that stimulation with 17β - estradiol leads to accumulation of KLF4 proteins, whereas inhibition of estrogen signals by tamoxifen and fulvestrant (ICI 182780) results in KLF4 degradation by VHL [12]. In addition, we have observed the KLF4 expression levels in various breast cancer cells is related to endocrine resistance.

In the present study, we have dissected the role of KLF4 in endocrine resistance and confirmed that the abnormal accumulation of KLF4 protein abundance in ER-positive breast cancer is a critical factor orchestrating tamoxifen resistance. We have further uncovered the upstream mechanism that determines the balance of KLF4 protein levels and unveiled an important role for Src-VHL cascade in governing KLF4 protein stability. Blockade of Src kinase activity could modulate KLF4 protein abundance through regulating VHL protein stability, which provides a novel strategy to overcome endocrine resistance.

2. Materials and methods

2.1. Cell Lines and Cell Culture

HEK293T, MCF7 and T47D were obtained from the American Type Culture Collection (Manassas, VA). The KLF4^+/+MEF and KLF4^−/− MEF were gifts from Dr. Engda Hagos (Colgate University). The Phoenix-A viral packaging cell line was a gift from Edward V. Prochownik (University of Pittsburgh). HEK293T, MCF7, T47D, and Phoenix-A were maintained in DMEM supplemented with 5 or 10% FBS, 1x antibiotic/antimycotic solution (100 units/ml streptomycin and 100 units/ml penicillin) (all from Invitrogen). The tamoxifen-resistant cell line HTR was a gift from Dr. David R. Gius (Northwestern University). The HTR cells were established as previously reported [24]. MCF7 was continuously treated with gradually increasing concentrations of tamoxifen ranging from 10⁻⁹ M to 10⁻⁶ M in around 40 weeks, and finally cultured continuously in the presence of 10⁻⁶ M tamoxifen. HTR was maintained with DMEM supplemented with 1μM tamoxifen (Sigma). MCF7-HER2 and MCF7-EGFR are MCF7 cells with stabilized expression of HER2 and EGFR gene. MCF7-HER2 and MCF7-EGFR were maintained with DMEM supplemented with 500ug/ml G-418 (Sigma). For estrogen stimulation, MCF7 or T47D cells were cultured in phenol red-free DMEM supplemented with 5% charcoal-stripped FBS for 48 h. Then, 10 nM 17β-estradiol (Sigma) was added to treat the cells. For EGF stimulation, MCF7 or HTR cells were cultured in phenol red-free DMEM supplemented with 5% charcoal-stripped FBS for 48 h. Then, 10nM EGF (Sigma) was added to treat the cells.

2.2. Antibodies, Chemicals and Peptides

Specific antibodies against KLF4 and p-Tyr (PY20) were purchased from Santa Cruz Biotechnology. Src, p-Src⁴¹⁸ and VHL were purchased from Cell Signaling. FLAG antibody and M2 beads were purchased from Sigma. p-Ser/Thr specific antibody (Clone 22A) was purchased from BD Bioscience. HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies were purchased from Promega. Dasatinib (S1021) and the 422 anti-cancer compounds library was from SelleckChem (Houston, TA). Peptides TAT (GRKKRRQRRRPPQ) and TAT-KLF4 (371-420AAa, GRKKRRQRRRPPQPGSCMPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYTKSSHLKAHLRTH) were synthesized by SelleckChem (Houston, TA). Cycloheximide (CHX) and MG132 were purchased from sigma.

2.3. Plasmids, siRNA and Transfection

KLF4 and VHL constructs were generated by PCR amplification of the full-length and partial coding sequence of human KLF4 and VHL, respectively, and then, subsequent subcloning into mammalian expression vectors with FLAG-HA or HA tag. KLF4 construct with Lys-43 point mutation was constructed as previously described [12]. VHL constructs with Tyr-112, -175, and -185 site mutations were constructed as follows: (Y112A-forward) 5'-CGCCGCATCCACAGCGCCCGAGGTCACCTTTG-3'; (Y112A-reverse) 5'-CAAAGGTGACCTCGGGCGCTGTGGATGCGGCG-3'; (Y175A-forward 5'-CCTAGTCAAGCCTGAGAATGCCAGGAGACTGGACATCG-3'; (Y175A-reverse) 5'-CGATGTCCAGTCTCCTGGCATTCTCAGGCTTGACTAGG-3'; (Y185A forward) 5'-ACATCGTCAGGTCGCTCGCCGAAGATCTGGAAGACC-3'; (Y185A-reverse) 5'-GGTCTTCCAGATCTTCGGCGAGCGACCTGACGATGT-3'. pRetroSuper-KLF4 shRNA was a gift from Dr. Daniel S. Peeper, Netherlands Cancer Institute). pLKO.1-ShVHL-1# (TRCN0000010460) and pLKO.1-ShVHL-2# (TRCN0000010461) were purchased from Sigma. The siRNA sequences for Src are 5’-GUACAUGAGCAAGGGGAGU-3’ and 5’- CAAGAGCAAGCCCAAGGAU-3’. The control siRNA sequence is 5’-CUUACGCUGAGUACUUCGA-3’. For plasmids and siRNA transfection, cells were plated to form a 50–70% confluent culture. The HEK293T, MCF7 and HTR cells were transfected using Lipofectamine 2000 (Invitrogen).

2.4. Lentiviral and Retroviral Infection

pLenti6/V5-KLF4, pLenti6/V5-KLF4-K43R, pLenti6/V5-VHL, pLenti6/V5-VHL-Y185A, pLKO.1-ShVHL-1#, pLKO.1-ShVHL-2# and pLKO.1-KLF4-shRNA were co-transfected with pVSV-G, pRRE, and pRSV-REV into HEK293T or Phoenix-A cells. Lipofectamine 2000 (Invitrogen) was used. The packaged lentiviral or retroviral particles were collected, mixed with polybrene, and added into MCF7 or HTR target cells. The stable cell lines were established by culturing the cells in the medium containing antibiotic blasticidin (10μg/ml) or puromycin (2 μg/ml).

2.5. Western Blotting and Immunoprecipitation Assay

Cells were harvested and lysed in radioimmune precipitation assay lysis buffer (Upstate Biotechnology) containing protease inhibitor mixture (Sigma) or 1x SDS loading buffer. The protein concentration was determined using Bio-Rad protein assay reagent. Western blotting was performed using anti-KLF4 (Santa Cruz Biotechnology), Src (Cell Signaling), p-Src⁴¹⁸ (Cell Signaling), VHL (Cell Signaling), FLAG (Sigma), β-actin (Sigma), p-Tyr (PY20, Santa Cruz Biotechnology), p-Ser/Thr (Clone 22A, BD) and HRP-conjugated goat anti-mouse or anti-rabbit secondary antibody (Promega). Signals were detected with ECL reagents (Bio-Rad). Semi-quantification of data was performed using NIH Image J. For immunoprecipitation assay, cell lysate was incubated with anti-FLAG M2 gel (Sigma) or anti-KLF4 (Santa Cruz Biotechnology) antibody overnight at 4 °C on a rotator, followed by addition of protein A/G plus agarose (Pierce) to the reaction containing anti-KLF4 antibody for 2 h at 4 °C. After five washes with radioimmune precipitation assay lysis buffer supplemented with protease inhibitor mixture, complexes were released from the anti-FLAG M2 gel by boiling for 5 min in 2x SDS-PAGE loading buffer.

2.6. Ubiquitylation Assay

cell pellets were lysed in 2% SDS and 5 mM dithiothreitol and diluted into 1% Nonidet P-40 buffer. The final concentrations in the lysate used for anti-KLF4 (Santa Cruz Biotechnology) immunoprecipitation were 0.2% SDS, 0.5 mM dithiothreitol, 1% Nonidet P-40, 50 mM Tris, pH 8, 150 mM NaCl, 10 mM MgCl₂ and protease inhibitor cocktail. After five washes with radioimmune precipitation assay lysis buffer supplemented with protease inhibitor mixture, complexes were released from the protein A/G plus agarose by boiling for 5 min in 2x SDS-PAGE loading buffer. The eluted samples were running through 8% SDS-PAGE gel and transferred to 0.2 μM nitrocellulose membrane and then blotting with ubiquitin antibody (P4D1, Cell signaling).

2.7. Cell Viability Assay

2×l0³ MCF7 or HTR cells were seeded into a 96-well plate and cultured overnight. Then, cells were treated with tamoxifen or Dasatinib for 96 h. Cell viability was determined using the CCK-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol.

2.8. Mammosphere growth assay

For mammosphere culture from MCF7 and HTR cells, cells were suspended at 1×10⁵ cells/ml and seeded into ultralow attachment plates (Corning, NY, USA) in serum-free DMEM/F12 (1:1) supplemented with 10 ng/ml basic fibroblast growth factor (b-FGF, Sigma), 20 ng/mL epidermal growth factor (EGF, Sigma), 10 μg/ml Insulin (Sigma), 0.5μg/ml hydrocortisone (Sigma), 0.4% BSA (Sigma), and 1x B27 (Invitrogen). Two milliliters of fresh media were added to each well every two days (without removing the old media). Cells grown in these conditions as nonadherent spherical clusters of cells were collected every seven days by gentle centrifugation and counted in culture with a Lionheart FX Automated Microscope (Biotek). For mammosphere viability assay, the spheres collected from two weeks’ culture were added into 96 well ultralow attachment plates (Corning, NY, USA) and treated with tamoxifen for one week. Then, mammosphere cell viability was determined using the CCK-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol.

2.9. Immunohistochemical Staining and immunofluorescence

Breast cancer Tissue array BC081120c sections of human breast cancer and adjacent tissues were purchased from Us Biomax Inc. The tissue array was placed in xylene for deparaffinization and ethyl alcohol for rehydration. The specific steps of deparaffinization and rehydration were as follows: Xylene I and II for 30 min; 100% ethanol I and II for 10 min; 95% ethanol for 10 min; 90% ethanol for 10 min; 80% ethanol for 10 min; and 70% ethanol for 10 min. After endogenous peroxidase activity was quenched with 3% H₂O₂ for 30 minutes, the antigen was retrieved by microwaving in citrate acid buffer (Darko). The sections were then incubated with primary antibody against KLF4 (1:100, sc-20691; SantaCruz) at 4 °C overnight. The validation of KLF4 antibody for immunohistochemical staining has been reported previous [11-13, 25-27]. After PBS washes, sections were incubated with biotinylated secondary antibodies and ABC kit Vectastain PK-6100 from Vector Labs. DAB (DAKO) was used to visualize the reaction, followed by counterstaining with Hematoxylin. After immunostaining, the sections were scanned and imaged by a single investigator who was not informed of the clinical characteristics. The tissue array was digitalized and evaluated on an ImageScope software (Aperio Technologies) using the positive pixel counting algorithm as previously reported [28]. This algorithm measures the percentage of DAB positive staining by area and the average intensity of positive staining. For immunofluorescence, cells expressing GFP-KLF4 were automatically captured and quantified by Lionheart FX Automated Microscope (Biotek).

2.10. Kaplan-Meier analysis

This analysis was performed with the online Kaplan-Meier Plotter tool (https://kmplot.com) for breast cancer [29]. Patients were divided according to best cutoff value. Probe 221841_s_at was used for KLF4. Post-progression survival was calculated and tested by the log-rank test.

2.11. Statistical analysis

The significance of the differences in the assays was analyzed by either Student’s t-test, or one-, or two-way ANOVA, followed by Tukey’s multiple comparison test. A value of p< 0.05 was considered significant.

3. Results

3.1. Abnormal accumulation of KLF4 in breast cancer

Recent studies showed the correlation of abnormal KLF4 protein accumulation with poor breast cancer prognosis and involvement of KLF4 in mediating estrogen signaling pathways [6, 7, 11-13]. However, the accumulation of KLF4 still seems controversial and it is still unknown how KLF4 accumulation is associated with various subtypes of breast cancers, including luminal A, luminal B, triple negative/basal-like, and HER2 types [30, 31]. To determine the KLF4 protein levels in different breast cancer molecular subtypes, we measured protein expression levels of KLF4 in various types of breast cancer cells as well as in a human breast tumor tissue array containing different subtypes of breast cancer. As shown in Figure 1A, while KLF4 expression is relatively low in mammary epithelial cell lines MCF10A and MCF12A and moderate in HER2 positive breast cancer cell lines SKBR3 and BT474, a significant accumulation of KLF4 is observed in triple negative and ER positive types of breast cancer cell lines MCF7, MDA-MB-468, MDA-MB-436 and HCC1937. We examined the expression of KLF4 in various types of breast cancer using a tissue array BC081120c containing 10 adjacent normal breast tissue (normal), 62 ER/PR positive breast cancer (ER⁺/PR⁺), 19 HER2 positive breast cancer (HER2) and 23 triple negative breast cancer (TNBC) specimens. The whole section staining showed the accumulation of KLF4 protein in breast cancer tissues in comparison with normal adjacent tissue (Figure 1B-C). In addition, among three molecular subtypes of breast cancers, we observed that KLF4 is significantly accumulated in ER/PR positive and triple negative breast cancer tissues (Figure 1C). Moreover, a Kaplan-Meier survival analysis showed that the high expression of KLF4 was associated with poor survival probabilities in overall breast cancer patients, ER positive breast cancer patients as well as patients with endocrine therapy (Figure 1D-F). In conclusion, abnormal accumulation of KLF4 in breast cancer predicts a poor prognosis in breast cancer patients.

Figure 1.

The abnormal accumulation of KLF4 in breast cancer. (A) Expression of KLF4 in mammary gland epithelial cells and various types of breast cancer cell lines. Actin serves as loading control. (B) Tissue array containing 10 adjacent normal breast tissues (normal), 62 ER/PR positive (ER⁺/PR⁺), 19 HER2 positive (HER2) and 23 triple negative (TNBC) breast cancer specimens were subjected to immunohistochemistry with anti-KLF4 and visualized by DAB staining. Representative images of normal and cancer tissue staining are shown. (C) Quantification of B. The asterisk indicates statistical significance between groups (p<0.05); n.s, not significant. (D-F) Kaplan-Meier survival assay of prognosis of KLF4 expression in overall breast cancer patient (D), ER positive breast cancer patient (E) and patient with endocrine therapy (F).

3.2. Identification of Dasatinib, a Src inhibitor, in promoting KLF4 protein ubiquitination and degradation based on screening of anti-cancer compound library

Previous studies suggested that KLF4 can be a potential target for ER/PR positive and triple negative breast cancers [7, 12, 32]. Thus, we initiated a search for possible drugs that could suppress KLF4 for potential therapy. We conducted a nonbiased screening based on an anti-cancer compound library containing 422 compounds under clinical trials using GFP-KLF4 signal as a readout [33]. For screening, we generated MDA-MB-231 cells with stable expression of the GFP-KLF4 and evaluated the inhibitory effects of 422 anti-cancer compounds on KLF4 expression levels (Figure 2A-E). As shown in Figure 2C-E, Dasatinib (Src inhibitor) was identified to be one of the best compounds in inhibiting KLF4 expression. The screening results were further confirmed by measuring KLF4 protein levels in response to Dasatinib using Western blotting (Figure 2E). Furthermore, Dasatinib significantly decreased KLF4 protein levels in MDA-MB-231, MCF7 and MCF7-HTR (tamoxifen resistant cells from MCF7, HTR) cells in dose-dependent manner (Figure 2F). At the same time, we observed no obvious alteration of KLF4 mRNA levels in MCF7 and HTR in response to different doses of Dasatinib (Figure 2G). Finally, the KLF4 ubiquitination assay showed that Dasatinib induced KLF4 protein ubiquitination (Figure 2H). These results suggest that Dasatinib, a Src inhibitor, is a potent compound that induces KLF4 protein ubiquitination and degradation.

Figure 2.

Screening of anti-cancer compound library identified that Dasatinib promotes KLF4 protein ubiquitination and degradation. (A) Established stable pLenti-GFP-N2 and pLenti-GFP-KLF4 expression in MDA-MB-231 cell line. (B) Representative images showing that major cellular localization of GFP-KLF4 is in nucleus. The expression of GFP-KLF4 was viewed by 60x fold microscopy. Scale bars, 5μm. (C) The plotted map represents the GFP-KLF4 fluorescence intensity levels normalized to DAPI in MDA-MB-231 cells after treatment with anti-cancer compounds. All MDA-MB-231 cells with pLenti-GFP-KLF4 stable expression were treated with an anti-cancer compound (10 μM) from 422 anti-cancer compound library for 24 hours and then the intensity of GFP and DAPI fluorescence was analyzed. Each dot represents one anti-cancer compound. The top 3 anti-cancer compounds are listed. (D) Representative images of GFP-KLF4 expression in MDA-MB-231 cells after treatment with Dasatinib for 24h during the anti-cancer compound screening. Scale bars, 100 μm. (E) Immunoblot analysis of KLF4 protein levels after treatment with indicated compounds at 10 μM for 24h. (F) Dose-dependent effect of Dasatinib on KLF4 expression in MDA-MB-231, MCF7 and MCF7-HTR (tamoxifen resistant cells, HTR) cells. Cells were treated with Dasatinib at indicated doses for 24h. (G) Relative KLF4 mRNA expression levels in MCF7 and HTR cells treated with Dasatinib at indicated doses for 24h. n.s, not significant. (H) Analysis of KLF4 ubiquitination in HTR cells treated with Dasatinib at indicated doses for 24h and then treated with 20 μM MG132 for 6h.

3.3. Aberrant KLF4 protein accumulation contributes to endocrine resistance

Previously, we demonstrated that the accumulation of KLF4 is regulated by estrogen signaling [12]. Accumulation of KLF4 protein in ER/PR positive breast cancer further suggested that KLF4 can be involved in cellular response to endocrine therapy. Thus, we measured KLF4 protein expression levels in several endocrine resistance cell lines derived from MCF7, including HTR, MCF7-EGFR, and MCF7-HER2. Interestingly, we found that KLF4 protein but not KLF4 mRNA levels were dramatically elevated in all three endocrine resistance cell lines (Figure 3A-B). In addition, we observed less KLF4 ubiquitination in HTR cells compared to MCF7 cells, which indicated that KLF4 is more stable in HTR cells (Figure 3C). To further confirm that KLF4 expression correlates with endocrine resistance, we modulated KLF4 expression in MCF7 by shRNA knockdown as well as overexpression. As shown in Figure 3D-E, KLF4 knockdown in HTR cells abrogated their resistance to tamoxifen (Figure 3E). Furthermore, overexpression of both wild-type (KLF4^WT) and degradation-resistance mutant (KLF4^K43R) KLF4 led to increased cell viability when MCF7 cells were treated with tamoxifen (Figure 3F-G). Moreover, MCF7 cells expressing KLF4 degradation-resistant mutant showed increased resistance to higher concentration (5μM, 10μM tamoxifen) of tamoxifen compared to cells expressing wild-type KLF4 (Figure 3F-G). These results suggested that the accumulation of KLF4 in ER-positive breast cancer can contribute to endocrine resistance.

Figure 3.

Aberrant levels of KLF4 protein contributes to endocrine resistance. (A) Expression of KLF4, EGFR and HER2 in MCF7 and its derivates cell lines MCF7-HTR (tamoxifen resistant cells, HTR), MCF7-HER2 (overexpression of HER2) and MCF7-EGFR (overexpression of EGFR). (B) Relative KLF4 mRNA expression levels in MCF7, HTR, MCF7-HER2 and MCF7-EGFR. n.s, not significant. (C) KLF4 ubiquitination in MCF7 and HTR cells treated with vehicle or 20 μM MG132 for 6h. (D-E) KLF4 knockdown in HTR cells sensitized them to tamoxifen. (D) Validation of KLF4 knockdown in MCF7 and HTR cells using Western blot. Actin was used as loading control. (E) For relative cell growth measurements, MCF7-ShScr, HTR-ShScr, MCF7-ShKLF4 and HTR-ShKLF4 were treated with different doses of tamoxifen ranging from 0.1 μM to 10 μM for 96h and then cell number was measured by CCK8 assay. The asterisk indicates statistical significance between ShKLF4 and the relative ShScr control group (p<0.05). (F-G) Overexpression of non-degradable KLF4 in MCF7 resulted in increased resistance to tamoxifen. (F) Expression of exogenous KLF4 wild-type (KLF4^WT) or non-degradable mutant (KLF4^K43R) in MCF7 cells. (G) For relative cell growth measurements, MCF7 cells overexpressing wildtype (KLF4^WT) or non-degradable mutant (KLF4^K43R) KLF4 and their control (Vector) were treated with different doses of tamoxifen ranging from 0.1 μM to 10 μM for 96h and then cell number was measured by CCK8 assay. The asterisk indicates statistical significance between wildtype (KLF4^WT) and non-degradable mutant (KLF4^K43R) KLF4 groups (p<0.05).

The accumulation of KLF4 protein but not mRNA in endocrine resistance cell lines indicated that the posttranslational modification of KLF4 is aberrant in endocrine resistance cells. To dissect the aberrant protein regulation, we monitored KLF4 protein stability under various conditions. In MCF7 cells, tamoxifen treatment dramatically decreased KLF4 protein half-life (Figure 4A-B). Furthermore, we observed that tamoxifen induces accumulation of KLF4 poly-ubiquitin conjugates in MCF7 cells, indicating that the KLF4 protein proteolysis is altered in response to endocrine treatment (Figure 4C). In addition, we observed that while ectopically expressed wild-type KLF4 undergoes degradation in response to tamoxifen or Fluvastatin (ICI), degradation-deficient mutant KLF4^K43R [34] failed to be degraded in response to tamoxifen or Fluvastatin (Figure 4D). This indicated that tamoxifen-induced KLF4 downregulation through increasing its ubiquitination and degradation. Nevertheless, the KLF4 protein is much more stable in HTR compared to MCF7 cells after treatment with tamoxifen (Figure 4E-F). Moreover, MCF7-EGFR cells with ectopic expression of EGFR also showed endocrine resistance in response to tamoxifen treatment. Similar to the estrogen 17-β-estradiol (E2) responses, cellular stimulation with growth factor EGF dramatically induced KLF4 accumulation, suggesting that enhanced EGFR-driven signaling in MCF7-EGFR could lead to KLF4 accumulation (Figure 4G). We also observed that KLF4 is more stable in MCF7-EGFR compared to MCF7 cells (Figure 4H). Finally, the Src inhibitor Dasatinib, which decreased KLF4 stability, abrogated estrogen induced KLF4 accumulation (Figure 4I). Taken together, the above results indicated that KLF4 protein is more stable in endocrine resistance cells such as HTR and MCF7-EGFR, which leads to KLF4 accumulation and cellular irresponsiveness to tamoxifen.

Figure 4.

Aberrant KLF4 protein turnover contributes to endocrine resistance. (A-B) Tamoxifen treatment decreased KLF4 protein half-life in MCF7 cells. (B) Quantification of results represented in (A). The density of KLF4 bands was quantified and normalized to the internal β-actin loading control. Cells were treated with CHX (100 μg/ml) at indicated time. The asterisk indicates statistical significance between groups (p<0.05). (C) KLF4 ubiquitination in MCF7 cells treated with tamoxifen at indicated doses for 24h and 20 μM MG132 for 6h. (D) Non-degradable mutant (KLF4^K43R) KLF4 shows resistance to tamoxifen and ICI induced decrease in comparison with KLF4 wildtype (KLF4^WT). MCF7 cells with overexpressed KLF4^WTor KLF4^K43R were treated with 1 μM ICI or tamoxifen for 6h and then subjected to western blotting for detection of F/H-KLF4 expression levels. Down panel shows the quantification results of up panel. (E-F) The half-life of KLF4 was increased in tamoxifen-treated HTR cells compared to MCF7 cells. MCF7 or HTR cells were pretreated with 1 μM tamoxifen for 4h and then pulse-chase with 100 μg/ml CHX for indicated hours. (F) Quantification of results represented in (E). The density of KLF4 bands was quantified and normalized to the internal β-actin loading control. The asterisk indicates statistical significance between groups (p<0.05). (G) MCF7 cells were starved for 24h and then stimulated with 10 nM E2 or 10 ng/ml EGF for the indicated time, and then subjected to western blotting for detection of KLF4 expression levels. (H) MCF7-EGFR and MCF7 cells were treated with 100 μg/ml CHX for the indicated time and then the expression of KLF4 and EGFR was detected by western blot. (I) Dasatinib abrogated estrogen (E2)-induced KLF4 accumulation. Cells were pretreated with 10 nM E2 and 1 μM Dasatinib for 6h before subjected to western blotting.

3.4. Src-mediated VHL phosphorylation orchestrates KLF4 stabilization in endocrine resistance cells

Next, we investigated the underlying mechanisms governing VHL-mediated KLF4 degradation in endocrine resistance. Recently, it has been reported that Src-mediated phosphorylation required for destabilization of VHL [35], suggesting that Src could participate in the VHL-KLF4 cascade in tamoxifen resistance. Src knockdown led to increased VHL and decreased KLF4 expression in both HTR and MCF7 cells (Figure 5A). Interestingly, Src kinase activity (p-Src⁴¹⁸) is also dramatically increased in HTR compared to MCF7 cells. Meanwhile, Src knockdown sensitized HTR cells to tamoxifen response (Figure 5B). In HTR cells, Dasatinib treatment increased VHL and decreased KLF4 expression (Figure 5C). In addition, while KLF4 remains stable, VHL is downregulated in HTR in the presence of tamoxifen and ICI (Figure 5D). Therefore, in HTR cells, an increase of Src kinase activity and a decrease of VHL protein expression contributed to the accumulation and stabilization of KLF4 protein in response to tamoxifen and ICI treatment. To further dissect the role of VHL in tamoxifen response, KLF4 ubiquitination and stabilization, we generated MCF7 cells with VHL knockdown. A decrease of VHL protein expression led to cell proliferation resistance to tamoxifen as well as KLF4 accumulation most likely as a result of the decrease of KLF4 ubiquitination (Figure 5E-G). Therefore, Src-mediated VHL regulation orchestrates KLF4 stabilization and thus contributes to tamoxifen resistance.

Figure 5.

Src-mediated VHL regulation orchestrates KLF4 stabilization. (A) Src knockdown led to increased VHL and decreased KLF4 expression in both MCF7 and HTR cells. For Src knockdown, MCF7 and HTR cells were transfected with Src siRNA. For detection of Src, VHL, p-Src⁴¹⁸, and KLF4 expression levels by western blot, cells were harvested at 72h after transfection. (B) MCF7 and HTR cells with siSrc or siScr expression were treated with different doses of tamoxifen ranging from 0.1 μM to 10 μM for 96h and then the number of cells was determined by CCK8 assay. The asterisk and octothorpe indicate statistical significance between siSrc and siScr groups in MCF7 and HTR cells respectively (p<0.05). (C) Dasatinib treatment led to increased VHL and decreased KLF4 expression in HTR cells. HTR cells were treated with Dasatinib at indicated doses for 24h and the expression of KLF4, p-Src⁴¹⁸, Src and VHL were detected by western blot. (D) VHL protein expression was upregulated in response to tamoxifen and ICI in both MCF7 and HTR cells. HTR and MCF7 cells were treated with 1 μM ICI or tamoxifen for 6h and then the expression of KLF4, VHL, Src and p-Src⁴¹⁸ were detected by immunoblotting. (E) MCF7 cells with ShVHL-1#, ShVHL-2# or ShScr expression were lysed and the expression of KLF4 and VHL were detected by western blot. β-actin serves as a loading control. (F). MCF7 cells with ShVHL-1#, ShVHL-2# or ShScr expression were treated with different doses of tamoxifen ranging from 0.1 μM to 10 μM for 96h and then the number of cells was determined by CCK8 assay. The asterisk indicates statistical significance between ShScr and ShVHL-1#, ShVHL-2# groups (p<0.05). (G) KLF4 ubiquitination in MCF7 cells 72h after transfection with VHL siRNA. Knockdown VHL decreased KLF4 ubiquitination in MCF7 cells.

To further explore how Src orchestrates the regulation of the VHL-KLF4 axis, we measured serine/threonine or tyrosine phosphorylation on VHL in the presence and absence of tamoxifen in both MCF7 and HTR cells. As shown in Figure 6A, we observed a decrease in serine/threonine phosphorylation and an increase in tyrosine phosphorylation in HTR compared to MCF7 cells. PhosphoSitePlus database (http://www.phosphosite.org) analysis reviled that there are three tyrosine phosphorylation sites, Y112, Y175 and Y185, on human VHL protein. The Y112 and Y175 were reported to be phosphorylated by Nek1 [36], while Y185 was linked to Src kinase [35] (Figure 6B). Thus, we decided to determine the involvement of these three phosphorylation sites in tamoxifen-mediated VHL phosphorylation. To determine the exact phosphorylation site which is involved in tamoxifen induced VHL phosphorylation, we engineered a set of point mutations in these three tyrosine residues (Figure 6B). We replaced three tyrosine residues individually by alanine on VHL (Y112A, Y175A, and Y185A) and then tested the effect of three mutations on VHL protein stability using IP-Western analysis. As shown in Figure 6C, while Y185A mutant abrogated both vehicle and tamoxifen induced tyrosine phosphorylation on VHL, Y112A and Y175A mutants had either no or very little effects, suggesting that 185 tyrosine is the major residue in mediating tamoxifen-induced phosphorylation of VHL. In addition, we observed that blockade of Src activity by using Src kinase-specific inhibitor Dasatinib led to a dramatic decrease in phosphorylation of wild-type but not Y185A mutant VHL (Figure 6D). Furthermore, in comparison with wild-type VHL, Y185A VHL mutant showed higher ability to increase KLF4 ubiquitination and downregulation in HTR cells as well as to sensitize tamoxifen in HTR cells (Figure 6E-G). Altogether, these results indicated that Src-mediated VHL phosphorylation plays an important role in regulating KLF4 protein stability in endocrine resistance cells.

Figure 6.

Phosphorylation of Tyrosine 185 on VHL contributes to VHL expression, KLF4 stabilization and tamoxifen resistance. (A) The tyrosine phosphorylation on VHL was increased in HTR compared to MCF7 cells. VHL was pulled down and the tyrosine or serine/threonine phosphorylation was detected. Right panel shows the quantification results of left panel. The asterisk indicates statistical significance between MCF7 and HTR cells from triplicate repeated experiments. (B) Schematic diagram of VHL tyrosine phosphorylation. VHL bears three potential tyrosine phosphorylation sites: Y112, Y175, and Y185. Strategy for engineering point mutation Y112A, Y175A and Y185A were indicated. (C) Replacement of tyrosine 185 by alanine on VHL abrogated tyrosine phosphorylation on VHL. F/H-VHL with point mutation at Y112, Y175 and Y185 were transfected into MCF7 cells and then immunoprecipitated with FLAG antibody followed by Western blotting using antibody against phosphor tyrosine. (D) Src kinase specific inhibitor Dasatinib dramatically decreased tyrosine phosphorylation on wildtype but not Y185A mutant VHL. Cells were pretreated with 1 μM Dasatinib for 6h before immunoprecipitated with FLAG antibody. (E) Y185A VHL led to more potency to decrease KLF4 expression in both MCF7 and HTR cells. (F) KLF4 ubiquitination in HTR cells with wildtype or Y185A mutant VHL transfection after 72h. (G) Y185A VHL showed more potency to sensitize tamoxifen. HTR cells with wild-type or Y185A mutant VHL were treated with tamoxifen ranging from 0.1 μM to 10 μM for 96h and then the number of cells was determined by CCK8. The asterisk indicates statistical significance between wildtype and Y185A mutant VHL groups in HTR cells (p<0.05). (H) Schematic diagram of VHL-mediated ubiquitination and PRMT5-mediated methylation in control of KLF4 stabilization. PRMT5-mediated methylation prevented VHL-mediated ubiquitination. Based on the methylation modification motif on KLF4, two peptides were synthesized: TAT-KLF4 (371-420Aa) and TAT. (I) KLF4 ubiquitination in HTR cells treated with 100 μg/ml TAT and TAT-KLF4 for 24h. (J) HTR and MCF7 cells were treated with 100 μg/ml TAT or TAT-KLF4 and tamoxifen at indicated concentrations, and the numbers of cells were determined by CCK8 assay. The asterisk indicates statistical significance between TAT and TAT-KLF4 in HTR cells (p<0.05).

Our previous work showed that PRMT5-mediated KLF4 triple arginine methylation inhibited VHL-mediated KLF4 ubiquitination and degradation [13]. To enhance VHL-mediated KLF4 ubiquitination, we synthesized TAT-KLF4 peptides, which cover the methylation motif of KLF4 (371-420Aa) and contains the cell-penetrating peptide TAT (Figure 6H). Treatment with TAT-KLF4 peptides significantly promoted KLF4 ubiquitination and decreased KLF4 protein expression (Figure 6I). Interestingly, TAT-KLF4 could significantly re-sensitize HTR cells to tamoxifen and slightly enhance MCF7 sensitivity (Figure 6J). These results indicated that an increase in VHL-mediated KLF4 ubiquitination could re-sensitize endocrine resistance cells.

3.5. Blockade of Src could re-sensitize endocrine resistance through modulating Src-VHL-KLF4 axis

Given that the Src-VHL cascade is critical in regulation of KLF4 accumulation in endocrine resistance, we then asked whether the Src-VHL-KLF4 cascade contributes to the endocrine resistance. We conducted pharmacological inhibition experiment and observed that Dasatinib abrogated tamoxifen resistance in HTR cells in cell proliferation assay, showing similar tamoxifen response as that of MCF7 cells (Figure 7A & B). KLF4 is one of the most critical factors for stem cell maintenance and differentiation as well as regulating breast cancer stem cell (CSC) maintenance and metastasis [7, 37]. Tamoxifen resistant cells showed enhanced mammosphere forming capacity compared to tamoxifen sensitive cells, suggesting an increased CSC population in tamoxifen resistant cells [38]. Thus, we analyzed the role of the Src-VHL-KLF4 cascade in endocrine therapy response by using the mammosphere assay. As shown in Figure 7C & D, HTR cells showed an increased ability of mammosphere formation compared to MCF7 cells. KLF4 knockdown decreased the number and the growth of mammospheres in both MCF7 and HTR cells in response to tamoxifen (Figure 7E-G). While the overexpression of either wild-type or VHL degradation-resistant mutant KLF4 increased the number of mammospheres, expression of KLF4 mutant showed higher potency, which indicated a critical role of KLF4 in promoting cancer stem cell maintenance (Figure 7H). The mammosphere viability assay showed strong correlation between KLF4 protein abundance and tamoxifen resistance (Figure 7H). While elevated expression of VHL led to a decrease in KLF4 expression and mammosphere formation, expression of Y185A mutant led to an increase in VHL accumulation, reduction in KLF4 expression and suppression of mammosphere formation compared to that of wild-type VHL, indicating that VHL inhibited cancer stem cell maintenance (Figure 7I-K). The results from the mammosphere viability assay further indicated that increased VHL phosphorylation results in the decreased tamoxifen sensitivity. Finally, blockade of the Src kinase activity by Dasatinib led to a decrease in mammosphere formation, which in turn sensitized cells to tamoxifen (Figure 7L-M). These results indicated that the Src-VHL-KLF4 cascade plays a critical role in regulating KLF4 abundance in endocrine resistance, which could serve as a potential target that overcomes endocrine resistance in breast cancer.

Figure 7.

Blockade of Src re-sensitizes endocrine resistance through modulation of the Src-VHL-KLF4 axis. (A-B) Dasatinib abrogated the tamoxifen resistance in HTR cells. (A) Expression of KLF4 in MCF7 and HTR cells treated with 1 μM Dasatinib and 1μM tamoxifen alone or combined for 24h. (B) Dose-dependent response of cells to tamoxifen in combination with 1μM Dasatinib. The asterisk and octothorpe indicate statistical significance between Dasatinib and vehicle treatment groups in both HTR and MCF7 cells respectively (p<0.05). (C-D) HTR showed significantly higher capability to form mammospheres compared to MCF7 cells. (C) Representative pictures of mammospheres. (D) Quantitative analysis of mammosphere number per 1×10³ MCF7 and HTR cells. The asterisk indicates statistical significance between MCF7 and HTR cells (p<0.05). (E) The schematic flow chart of mammosphere growth assay. (F-G) KLF4 knockdown decreased number of mammospheres in both MCF7 and HTR cells and mammosphere growth tamoxifen resistance only in HTR cells. (F) Representative pictures of mammospheres. (G) Growth of mammospheres under the treatment of tamoxifen at indicated concentrations. The asterisk indicates statistical significance between ShScr and ShKLF4 groups in HTR cells (p<0.05). (H) Overexpression of KLF4 (both KLF4 wild-type and K43R mutant) increased number of mammosphere clones compared to control Vector group. The asterisk indicates statistical significance between KLF4 wildtype and K43R mutant groups. The octothorpe indicates statistical significance between KLF4 wildtype and vector groups (p<0.05). (I-K) While elevated expression of wild-type VHL led to a decrease in mammosphere clones, expression of VHL Y185A mutant resulted in decreased mammosphere formation potency. (I) Expression of KLF4 and VHL in HTR cells with wildtype or Y185A mutant VHL. Expression of Y185A mutant VHL showed more decreased KLF4 accumulation. (J) Representative pictures of mammospheres. (K) Mammosphere growth under the treatment of tamoxifen at indicated concentrations. The asterisk indicates statistical significance between VHL wild-type and Y185A mutant groups in HTR cells (p<0.05). (L-M) Blockade of Src kinase activity by Dasatinib also led to decreased mammosphere formation that in turn sensitized tamoxifen sensitivity. (L) Representative pictures of mammospheres. (M) Dose-dependent response of HTR and MCF7 cells to tamoxifen 3D Matrigel culture. The asterisk and octothorpe indicate statistical significance between Dasatinib and vehicle treatment groups in both HTR and MCF7 cells respectively (p<0.05).

4. Discussion

Results from the present dissection of KLF4 have revealed its role in carcinogenesis and drug resistance through regulating cell cycle, apoptosis, genomic integrity, and signal transduction. Constitutive turnover of KLF4 is controlled by VHL-mediated ubiquitination and proteasomal degradation in response to DNA damage and estrogen signaling [12, 13]. However, previous work showed the presence of 6 splice variants of KLF4 [39], and that the KLF4 protein is tightly controlled by posttranslational modifications, such as ubiquitination and degradation by E3 ligase VHL [34], FBOX22 [15], APC/C-Cdh1 [40], and β-Trcp [41] in various conditions, highly suggesting the role of posttranslational modifications in regulating KLF4 in carcinogenesis. Here we reported that proteolytic regulation of KLF4 by VHL is important in endocrine signal transduction and endocrine resistance. Results from our biochemical and pathological analyses unveiled the importance of KLF4 abundance regulation by Src/VHL-mediated protein degradation pathway in governing endocrine resistance in ER-positive breast cancer, which is consistent with the previously observed impact of Src and VHL in endocrine resistance [42, 43]. In tamoxifen sensitive cells, treatment of tamoxifen or ICI disrupts the estrogen receptor activity and results in inactivation of Src kinase, VHL dephosphorylation, and KLF4 degradation, leading to inhibition of mammosphere growth and cell viability. In tamoxifen resistance cells, the growth factor signaling or other pathways consistently activate Src kinase and reduce the cellular response to tamoxifen treatment. The hyperactivated Src leads to phosphorylation of VHL that further results in the destruction of VHL and accumulation of KLF4, therefore promoting the mammosphere growth and cell viability but reducing cellular response to tamoxifen (Figure 8).

Figure 8.

Hypothetical working model. (left) In tamoxifen-responsive breast cancer cells, low Src activity results in accumulation of VHL leading to degradation and downregulation of KLF4. (middle) In tamoxifen-resistant breast cancer cells, hyperactivated Src due to crosstalk from HER2 and other EGFR signaling enhances degradation of VHL. A decrease in VHL levels results in stabilization of KLF4 leading to endocrine resistance. (right) Blockade of Src activity by Dasatinib prevents VHL degradation and downregulation. Increase in VHL levels promotes KLF4 degradation, thereby restoring the endocrine response for tamoxifen resistant breast cancer cells.

17-β-Estradiol, ERα, and its coregulators have been implicated in the mammary tumorigenesis and progression. Src kinase is known to be increased in breast cancers compared with normal breast tissue [44]. The reciprocal regulation of ERα and Src kinase medicated estrogen receptor-mediated gene transcription and mitogenesis, which contribute to mammary tumorigenesis and progression [22, 23]. Tamoxifen resistance after long-term treatment showed obvious increase in Src activity, which correlates with acquired mobility and invasive ability [19, 21]. Elevated Src kinase activity could attenuate tamoxifen response that is associated with poor prognosis in clinic [45]. In our cultured-cell model, we consistently observed the aberrant Src with high activity in tamoxifen resistant cell lines HTR and MCF7-EGFR cells. This over-activated Src kinase in HTR cells might be one of the reasons conferring cellular irresponsiveness to tamoxifen. Based on the screening of tyrosine phosphorylation sites on VHL in response to tamoxifen, we have identified that the 185 tyrosine is the critical modification residue in mediating Src regulated VHL protein stability, which is consistent with the recent report that Src could phosphorylate VHL and lead to ubiquitination and proteasome-mediated degradation of VHL [35]. Other than HTR and MCF7-EGFR cells, even in MCF7 cells, the estrogen 17-β-estradiol and EGF could directly stimulate Src kinase activity and subsequent VHL degradation and KLF4 accumulation. Therefore, the cascade Src kinase-VHL-KLF4 not only exists in endocrine resistance but also is critical for normal cell growth [46]. The Src kinase is also critical for pluripotent stem cells and cancer stem cell load [46, 47], where KLF4 plays a critical role in these processes. Our results indicated blockade of Src-VHL-KLF4 cascade by pharmacological Src inhibitor has a dramatic effect on mammosphere growth and viability, which further confirmed the impact of this signaling cascade in regulating pluripotent stem cells, expansion of cancer stem cell as well as endocrine resistance.

The potent orally available Src inhibitor Dasatinib was approved by the FDA for the treatment of chronic myelogenous leukemia [48]. The clinic trail using Dasatinib in combination with endocrine therapy has also been investigated, although the results remain controversial. In a Phase II Trial, Dasatinib with Fluvastatin or exemestane did not improve clinical benefits in progression-free survival (PFS) for women with metastatic BC [49]. In contrast, the combination of Dasatinib and letrozole did show improved PFS in women with ER+, HER2- metastatic BC [50]. Recent studies indicated the Dasatinib is only sensitive to tamoxifen resistance models consistent with our observation in the HTR model. It was shown that Dasatinib reduced effect in ER genomic activity, but not in the aromatase inhibitors resistance model (LTED) [51]. Other than the disruption of Src-VHL cascade by Dasatinib, we also showed enhance in VHL-mediated KLF4 degradation by peptide TAT-KLF4 (371-420AAa) which mimic the KLF4 methylation motif, which could also overcome tamoxifen resistance. We recently identified a novel PRMT5-KLF4 binding inhibitor WX2-43 [52]. Based on the results of TAT-KLF4 (371-420AAa), the PRMT5-KLF4 binding inhibitor WX2-43 might also have promising effects in overcoming endocrine resistance. Altogether, our results implicated controlling KLF4 abundance is a key during breast carcinogenesis and endocrine resistance. KLF4 expression levels might be an important marker for patient selection to gain maximum clinical benefit from combination therapy for Dasatinib as well as TAT-KLF4 (371-420AAa) and endocrine therapy. In conclusion, KLF4 plays a critical role in breast cancer initiation, progression, and endocrine resistance. Our results demonstrated the impact of the Src-VHL-KLF4 axis in endocrine resistance and further implicated a potential value of the Src-VHL-KLF4 axis in precision therapy, especially for patients who suffer from endocrine resistance with high KLF4 expression levels.

Highlights.

• Proteolytic regulation of KLF4 by ubiquitylation plays a role in breast cancer endocrine resistance

• Src-mediated VHL phosphorylation regulates VHL degradation that in turn determines KLF4 stabilization

• Modulating Src-VHL-KLF4 axis re-sensitizes endocrine resistance

• Enhancement of VHL-KLF4 ubiquitination by TAT-KLF4 (371-420AAa) peptides re-sensitizes endocrine resistance

Abbreviations

CHX

Cycloheximide

E2

17β-estradiol

ER

Estrogen receptor

ER⁺/PR⁺

Estrogen receptor /progestin receptor positive breast cancer

IHC

immunohistochemistry

KLF4

Krüppel-like factor 4

PFS

Progression-free survival

TNBC

Triple negative breast cancer