TAM-resistant Breast Cancer Research Articles

Solid lipid nanoparticles: Reversal of tamoxifen resistance in breast cancer

The objective of the present study was to investigate the effect of tamoxifen (Tam) loaded solid lipid nanoparticles (SLNs) on MCF7 Tam-resistant breast cancer cells (MCF7-TamR). Tam-SLNs were produced by the hot homogenization method. The characterization studies of Tam-SLNs demonstrated good physical stability with small particle size. The in vitro cytotoxicity results showed that Tam-SLNs enhanced the efficacy of Tam and reversed the acquired Tam resistance by inducing apoptosis, altering the expression levels of specific miRNA and the related apoptosis-associated target-genes in both MCF7 and MCF7-TamR cells without damaging the MCF10A control cells (p < 0.05). In conclusion, we demonstrated a molecular mechanism of the induction of apoptosis by Tam-SLNs in MCF7 and MCF7-TamR cells, and thus, we demonstrated that Tam-SLNs were more effective than Tam. The present study suggests that the use SLNs may be a potential therapeutic strategy to overcome Tam-resistance in breast cancer for clinical use.

Oncoprotein HBXIP enhances HOXB13 acetylation and co-activates HOXB13 to confer tamoxifen resistance in breast cancer

BackgroundResistance to tamoxifen (TAM) frequently occurs in the treatment of estrogen receptor positive (ER+) breast cancer. Accumulating evidences indicate that transcription factor HOXB13 is of great significance in TAM resistance. However, the regulation of HOXB13 in TAM-resistant breast cancer remains largely unexplored. Here, we were interested in the potential effect of HBXIP, an oncoprotein involved in the acceleration of cancer progression, on the modulation of HOXB13 in TAM resistance of breast cancer.MethodsThe Kaplan-Meier plotter cancer database and GEO dataset were used to analyze the association between HBXIP expression and relapse-free survival. The correlation of HBXIP and HOXB13 in ER+ breast cancer was assessed by human tissue microarray. Immunoblotting analysis, qRT-PCR assay, immunofluorescence staining, Co-IP assay, ChIP assay, luciferase reporter gene assay, cell viability assay, and colony formation assay were performed to explore the possible molecular mechanism by which HBXIP modulates HOXB13. Cell viability assay, xenograft assay, and immunohistochemistry staining analysis were utilized to evaluate the effect of the HBXIP/HOXB13 axis on the facilitation of TAM resistance in vitro and in vivo.ResultsThe analysis of the Kaplan-Meier plotter and the GEO dataset showed that mono-TAM-treated breast cancer patients with higher HBXIP expression levels had shorter relapse-free survivals than patients with lower HBXIP expression levels. Overexpression of HBXIP induced TAM resistance in ER+ breast cancer cells. The tissue microarray analysis revealed a positive association between the expression levels of HBXIP and HOXB13 in ER+ breast cancer patients. HBXIP elevated HOXB13 protein level in breast cancer cells. Mechanistically, HBXIP prevented chaperone-mediated autophagy (CMA)-dependent degradation of HOXB13 via enhancement of HOXB13 acetylation at the lysine 277 residue, causing the accumulation of HOXB13. Moreover, HBXIP was able to act as a co-activator of HOXB13 to stimulate interleukin (IL)-6 transcription in the promotion of TAM resistance. Interestingly, aspirin (ASA) suppressed the HBXIP/HOXB13 axis by decreasing HBXIP expression, overcoming TAM resistance in vitro and in vivo.ConclusionsOur study highlights that HBXIP enhances HOXB13 acetylation to prevent HOXB13 degradation and co-activates HOXB13 in the promotion of TAM resistance of breast cancer. Therapeutically, ASA can serve as a potential candidate for reversing TAM resistance by inhibiting HBXIP expression.

Differential microRNA expression profiles in tamoxifen-resistant human breast cancer cell lines induced by two methods.

Tamoxifen (TAM) resistance has become a severe problem for endocrine therapy of breast cancer. The present study investigated the association between microRNA (miRNA) expression and TAM resistance in breast cancer. The TAM-resistant breast cancer MCF-7C and MCF-7T cell lines were established using the human breast cancer cell line MCF-7 as the parental cell line and 4-hydroxytamoxifen (OHT) as the screening drug in vitro. The MCF-7C cell line was established by dose stepwise induction beginning with a low concentration of OHT; the MCF-7T cell line was established by temporal stepwise induction beginning with a high concentration of OHT. Differential miRNA expression profiles between TAM-sensitive (MCF-7) and TAM-resistant (MCF-7C and MCF-7T) breast cancer cell lines were detected and analyzed using RNA sequencing technology. The results of western blot analysis indicated that the level of ERα protein expression in drug-resistant cells was significantly increased. A total of 1,646 miRNAs were detected in all samples, including 1,376 known miRNAs and 270 predicted miRNAs. There were 118 miRNAs expressed at significantly different levels between MCF-7C and MCF-7 cells (P<0.05); among them, 67 miRNAs were upregulated (P<0.05) and 51 miRNA were downregulated (P<0.05). There were 42 miRNAs expressed at significantly different levels between MCF-7T and MCF-7 (P<0.05); among them, 23 miRNAs were upregulated (P<0.05) and 19 miRNAs were downregulated (P<0.05). There were 126 miRNAs with significant differences between MCF-7C and MCF-7T (P<0.05); among them, 76 miRNAs were upregulated (P<0.05) and 50 miRNAs were downregulated. On the basis of the results of the present study, we hypothesize that miR-21, miR-146a, miR-148a, miR-34a and miR-27a may serve important roles in mediating TAM resistance in breast cancer, and have potential as therapeutic targets for TAM-resistant breast cancer.

Establishment of an acquired tamoxifen-resistant human breast cancer cell line and its mechanism of 18F-FDG uptake reduction

Objective To establish the acquired tamoxifen (TAM)-resistant human breast cancer cell line T47D-TamR, compare the 18F-FDG uptake rate between T47D-TamR and its parental cell line T47D, and study the mechanism. Methods Long-term step-wise drug stimulation was used for cell line T47D-TamR establishment and then the cell proliferation and resistance index (RI) were determined by MTT assay. The 18F-FDG uptake rates of T47D-TamR and T47D cells were measured in the setting of different cell counts, reaction time, 18F-FDG dosages and glucose concentrations. The LDH activity, cellular ATP level and lactic acid concentration in cell supernatant of T47D-TamR and T47D cells were detected. Western blot was used to examine the expression of ERα, Glut-1, phosphorylated AMPK (p-AMPK) and phosphorylated mTOR (p-mTOR). Two-sample t test and two-way analysis of variance were used to analyze the data. Results T47D-TamR cell line was successfully established and its drug RI was 1.97±0.08, with a significantly decreased cell proliferation efficacy (F=230.10, P<0.05). Significant differences of 18F-FDG uptake rates were found between T47D-TamR cell and T47D cell when changing the cell count, reaction time, and 18F-FDG dosage (F values: 419.00-1 116.00, all P<0.05). The LDH activities of T47D cell and T47D-TamR cell were (0.42±0.04) and (0.32±0.02) U/mg protein, cellular ATP levels were (19.99±0.32) and (14.01±0.70) nmol/mg protein, lactic acid concentrations in cell supernatant were (2.95±0.05) and (2.02±0.07) mmol/L, respectively. The differences of above parameters between the two groups were significant (t values: 4.39-18.80, all P<0.05). The relative expressions of ERα, p-AMPK, p-mTOR, Glut-1 were 0.26±0.03, 0.36±0.06, 0.75±0.11, 0.35±0.07 in T47D cell, and 0.17±0.02, 0.61±0.09, 0.52±0.08, 0.21±0.04 in T47D-TamR cell, and there were significant differences (t values: 12.20-16.45, all P<0.05) between the two groups. Conclusions Compared with parental cells, T47D-TamR cells have lower 18F-FDG uptake rate, LDH activity, cellular ATP level and lactic acid concentration, increased p-AMPK expression and decreased ERα, p-mTOR, Glut-1 expression, indicating the decreased glycolysis ability in TAM-resistant breast cancer cells. Key words: Breast neoplasms; Tumor cells, cultured; Tamoxifen; Fluorine radioisotopes; Deoxyglucose

GPER promotes tamoxifen-resistance in ER+ breast cancer cells by reduced Bim proteins through MAPK/Erk-TRIM2 signaling axis.

Tamoxifen resistance is a major clinical challenge in breast cancer treatment. Our previous studies find that GPER and its down-stream signaling play a pivotal role in the development of tamoxifen (TAM) resistance. cDNA array analysis indicated a set of genes associated with cell apoptosis are aberrant in GPER activated and TAM-resistant MCF-7Rcells compared with TAM-sensitive MCF-7cells. Among these genes, Bim (also named BCL2-L11), a member of the BH3-only pro-apoptotic protein family is significantly decreased, and TRIM RING finger protein TRIM2 (a ubiquitin ligase) is highly expressed in MCF-7R. To understand the mechanism of TAM-resistance in GPER activated ER+ breast cancer, the function of TRIM2 and Bim inducing cell apoptosis was studied. By using immunohistochemical and western blot analysis, there is an adverse correlation between TRIM2 and Bim in TAM-resistant breast tumor tissues and MCF-7R cells. Knockdown Bim in TAM-sensitive MCF-7cells or overexpression of Bim in TAM-resistant MCF-7cells significantly changed its sensibility to TAM through altering the levels of cleaved PARP and caspase-3. Activation of GPER and its downstream signaling MAPK/ERK, not PI3K/AKT, led to enhanced TRIM2 protein levels and affected the binding between TRIM2 and Bim which resulted in a reduced Bim in TAM-resistant breast cancer cells. Thus, the present study provides a novel insight to TAM-resistance in ER-positive breast cancer cells.

The miR-29 transcriptome in endocrine-sensitive and resistant breast cancer cells

Aberrant microRNA expression contributes to breast cancer progression and endocrine resistance. We reported that although tamoxifen stimulated miR-29b-1/a transcription in tamoxifen (TAM)-resistant breast cancer cells, ectopic expression of miR-29b-1/a did not drive TAM-resistance in MCF-7 breast cancer cells. However, miR-29b-1/a overexpression significantly repressed TAM-resistant LCC9 cell proliferation, suggesting that miR-29b-1/a is not mediating TAM resistance but acts as a tumor suppressor in TAM-resistant cells. The target genes mediating this tumor suppressor activity were unknown. Here, we identify miR-29b-1 and miR-29a target transcripts in both MCF-7 and LCC9 cells. We find that miR-29b-1 and miR-29a regulate common and unique transcripts in each cell line. The cell-specific and common downregulated genes were characterized using the MetaCore Gene Ontology (GO) enrichment analysis algorithm. LCC9-sepecific miR-29b-1/a-regulated GO processes include oxidative phosphorylation, ATP metabolism, and apoptosis. Extracellular flux analysis of cells transfected with anti- or pre- miR-29a confirmed that miR-29a inhibits mitochondrial bioenergetics in LCC9 cells. qPCR,luciferase reporter assays, and western blot also verified the ATP synthase subunit genes ATP5G1 and ATPIF1 as bone fide miR29b-1/a targets. Our results suggest that miR-29 repression of TAM-resistant breast cancer cell proliferation is mediated in part through repression of genes important in mitochondrial bioenergetics.

Abstract 3428: Tamoxifen differentially regulates miR-29b-1 and miR-29a depending on endocrine sensitivity in breast cancer

Abstract Acquired endocrine-resistance occurs in ~ 40% of estrogen receptor α (ERα)+ breast cancer patients after tamoxifen (TAM) or aromatase inhibitor therapy. MicroRNAs (miRs) are dysregulated in breast cancer, but their contribution to endocrine-resistance is not yet completely understood. Microarray analysis of miRNAs in TAM-sensitive MCF-7 versus TAM-resistant LY2 breast cancer cells (derived from MCF-7 cells) identified differential regulation of miR-29b-1 and miR-29a. miR-29a and miR-29b-1, which are co-transcribed as a single primary-miR, were repressed by TAM in MCF-7 and stimulated by TAM in LY2 cells. We confirmed these observations by real time qPCR in MCF-7 and LY2 cells and observed that 4-OH-TAM (4-OHT) also downregulated miR-29b-1/a in LCC2 and LCC9 endocrine-resistant cells. These changes were at the primary transcriptional level. The role of these two miRs in endocrine-resistant breast cancer is unknown. We tested the hypothesis that the TAM-stimulated increase in miR-29b-1/a promotes endocrine-resistance by downregulating targets that contribute to TAM’s antiproliferative/pro-apoptotic activity. Our goals were to: 1) Determine if ERα was responsible for TAM regulation of miR-29b-1/a expression; 2) Determine the functional role(s) of miR-29b-1/a; 3) Identify specific miR-29b-1/a target genes that are differentially regulated by 4-OHT in MCF-7 versus LCC9 and LY2 cells. Knockdown of ERα blocked 4-OHT inhibition of miR-29b-1/a in MCF-7 and 4-OHT simulation of miR-29-b-1/a transcription in LCC9 and LY2 cells. Transient over-expression of miR-29b-1/a inhibited proliferation of MCF-7, LCC9, and LY2 cells and decreased migration and colony formation of LY2 cells. Repression of miR-29b-1/a has no significant effect on MCF-7, LCC9, or LY2 cell proliferation. Repression of miR-29-b-1/a did not sensitize the TAM-resistant cells to growth inhibition by 4-OHT. We observed that the bona fide miR-29 target gene, DICER1, was inhibited by 4-OHT in LY2 cells and anti-miR29b-1 or anti-miR-29a decreased the suppression of DICER1 by 4-OHT. These data suggest that 4-OHT-ERα increases miR-29b-1/a which target and downregulate DICER 1 in endocrine resistant breast cancer cells. Future studies will identify other targets of miR-29b-1/a to better understand the pathways leading to acquired endocrine resistance. Citation Format: Penn Muluhngwi, Carolyn M. Klinge. Tamoxifen differentially regulates miR-29b-1 and miR-29a depending on endocrine sensitivity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3428. doi:10.1158/1538-7445.AM2017-3428

Essential role of Notch4/STAT3 signaling in epithelial–mesenchymal transition of tamoxifen-resistant human breast cancer

We previously demonstrated that tamoxifen (TAM)-resistant human breast cancer (TAMR-MCF-7) cells showed increased expression of mesenchymal marker proteins compared to the parent MCF-7 cells. Notch is functionally important in the promotion of epithelial–mesenchymal transition (EMT) during both development and tumor progression. Notch1 and Notch4 have been reported as prognostic markers in human breast cancer. Here, we indicated that Notch4, but not Notch1, plays a critical role in the regulation of EMT signaling in TAMR-MCF-7 cells. Notch4 suppression by either Notch inhibitors or Notch4 siRNA attenuated EMT signaling. Tyrosine-phosphorylated STAT3 protein is known as a crucial signaling molecule in the regulation of tumorigenesis and metastasis. We found that TAMR-MCF-7 cells exhibited constitutive STAT3 phosphorylation, and Notch inhibition reduced the level of activated STAT3 in TAMR-MCF-7 cells. An intrasplenic injection model of liver metastases was performed using TAMR-MCF-7 cells. Mice injected with N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT, 10 mg/kg) formed smaller splenic tumors and showed a reduced micrometastatic tumor burden in their livers compared with the control group treated with vehicle. To conclude, Notch4 could be a potential target to prevent metastasis in TAM-resistant breast cancer.

THE EFFECT OF SOLID LIPID NANOPARTICLES ON TAMOXIFEN-RESISTANT BREAST CANCER CELLS

&lt;p class="lead"&gt;To overcome the acquired Tamoxifen (Tam) resistance in Tam-resistant breast cancer cells without damaging normal cells, we have examined the therapeutic efficacy of Tam-loaded solid lipid nanoparticles (SLNs). Tam-loaded SLNs were produced by hot homogenization method. After characterization, &lt;em&gt;in vitro&lt;/em&gt; cytotoxic and apoptotic activity of Tam-SLNs were evaluated in MCF7, MCF7-TamR (Tam-resistant breast cancer cells) and MCF10A cells. Tam-SLNs had an average size nearly 300 nm and a zeta potential of approximately-40 mV. &lt;em&gt;In vitro&lt;/em&gt; cytotoxicity results showed that Tam-SLNs indicated the cytotoxic and apoptotic activity on MCF7 and MCF7-TamR cells. We found that MCF7-TamR cell viability was also suppressed significantly by Tam-SLNs and thus, Tam-SLNs could delay and overcome Tam-resistance (p&amp;lt;0.05). Furthermore, the Tam-SLNs did not induce apoptosis on MCF10A control cells. The lowest MCF10A cell viability was 83.0% whereas MCF7 and MCF7-TamR (R↔ and R↑) cells viability are reduced to 21.98%, 27.5% and 29.4% at 10 µM of Tam-SLNs, respectively (p&amp;lt;0.05). The obtained results were supported by apoptosis assays. SLNs-delivery system provided therapeutic efficacy to overcome Tam-resistance thanks to unique features of SLNs including small size, drug accumulation in the tumor site and controlled drug release. Therefore, Tam-SLNs may have therapeutic potential for the treatment of TAM-resistant breast cancer.&lt;/p&gt;

Abstract 619: Selective estrogen mimics for the treatment of tamoxifen-resistant breast cancer

Abstract Endocrine-resistant breast cancer, whether de novo or acquired, is a major clinical obstacle. Although recent clinical trials have demonstrated the efficacy of 17β-estradiol (E2) or diethylstilbestrol (DES) following exhaustive use of antiestrogens, E2 treatment is associated with major side effects such as an increased risk of other gynecological cancers, deterring the clinical community from adopting it as a treatment strategy. Our lab has previously shown that Protein kinase C alpha (PKCα) overexpression predicts tamoxifen (TAM) resistance in the clinic (Tonetti DA et al., Br J Cancer. 2003) and may also predict a positive response to an estrogenic therapy (Chisamore MJ et al., Clinical Cancer Research. 2001). Further, the ectopic overexpression of PKCα in T47D breast cancer cells, led to a TAM-resistant, E2-inhibited phenotype in vivo which was accompanied by the translocation of estrogen receptor alpha (ERα) to extranuclear sites (Perez White B et al., Molecular Cancer. 2013). The purpose of this study was to identify novel selective estrogen mimics (SEMs), which could achieve the positive therapeutic effects of E2 treatment in TAM-resistant breast cancers, while minimizing the side effects. In vitro screening identified two SEMs, BTC and TTC-352, which displayed estrogenic activity in breast cancer cell lines. BTC and TTC-352 treatment resulted in significant tumor regression in two xenograft models of TAM-resistant, PKCα-overexpressing breast cancer. Similar to E2, T47D:A18/PKCα tumor regression was accompanied by translocation of ERα to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment, however, did not result in growth of parental, TAM-sensitive xenograft tumors. Endometrial thickening, caused by both E2 and TAM, is directly associated with gynecological carcinogenesis and uterine cancer. Interestingly, SEM treatment did not increase uterine weight in mice suggesting negligible hormonal stimulation in gynecological tissues. Both BTC and TTC-352 resulted in regression of two TAM-resistant breast cancer models, while displaying enhanced safety compared to E2 and TAM. These data suggest that further development of SEMs targeted to TAM-resistant breast cancer is a feasible therapeutic strategy. Citation Format: Mary Ellen Molloy, Bethany Perez White, Huiping Zhao, Bradley T. Michalsen, Hitisha K. Patel, Jiong Zhao, Rui Xiong, Marton I. Siklos, Gregory R.J. Thatcher, Debra A. Tonetti. Selective estrogen mimics for the treatment of tamoxifen-resistant breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 619. doi:10.1158/1538-7445.AM2014-619

Abstract 1634: A novel small molecule inhibitor of IRE1alpha reverses endocrine resistance in breast cancer cells

Abstract Over two-thirds of all breast cancer patients are estrogen receptor alpha positive (ER+). However, almost 50% of all ER+ breast tumors do not respond or respond and later fail on endocrine therapy. Resistance to endocrine therapy remains a significant clinical problem and advanced ER+ breast cancer is largely an incurable disease. The mechanisms of resistance to endocrine therapy remain unclear. We have reported that the unfolded protein response (UPR), a stress-induced cellular adaptive mechanism, acts as a switch controlling the balance between cell death and cell survival in response to these therapies. UPR is activated by three molecular sensors: IRE1alpha, ATF6, PERK. Several components of the UPR, including IRE1alpha, are up-regulated in endocrine resistant cells. We now report a novel small molecule inhibitor of IRE1alpha: N-(4-Phenoxy-phenyl)-2-(5-pyridin-3-yl-2H-[1,2,4]triazol-3-ylsulfanyl)-acetamide (NPPTA). We show that NPPTA interacts with IRE1alpha in vitro and can decrease IRE1alpha protein levels. NPPTA treatment alone inhibits cell proliferation in antiestrogen resistant ER+ breast cancer cells more effectively than in sensitive cells, and in a dose- and time-dependent manner. Normal breast epithelial cells are unaffected by NPPTA. In addition, NPPTA and TAM synergistically inhibit cell proliferation in TAM resistant breast cancer cells. NPPTA's activities are associated with induced G1 cell cycle arrest, increased apoptosis/necrosis, and inhibition of anti-apoptotic signaling. Targeting IRE1alpha may be a new and effective strategy to reverse endocrine resistance in breast cancer. Citation Format: Ayesha N. Shajahan-Haq, Jacqueline Smith, Siva Dakshanamurthy, Difei Wang, Ahreej E. Eltayeb, Milton L. Brown, Robert Clarke. A novel small molecule inhibitor of IRE1alpha reverses endocrine resistance in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1634. doi:10.1158/1538-7445.AM2014-1634

The effect of Shugan-Yishen formula on HER2-ERK/MAPK-ERα Pathway of TAM-resistant breast cancer cell lines

Objective To investigate the effect of Shugan-Yishen formula on Tamoxifen-resistant MCF-7 cell line（LCC9） through HER2-ERK/MAPK-ERα pathway. Methods Four serums were prepared with the method of serum pharmacology：the serum of Shugan-Yishen formula, Shugan-Yishen formula plus Tamoxifen（TAM）, blank and TAM alone. After LCC-9 cells were affected in the above-mentioed serums in vitro, the inhibition rate and the level of mRNA as well as the protein expression of HER2, ERα, ERK/MAPK, p-ERK/MAPK were detected. Results MTT showed that TAM alone group、Shugan-Yishen formula group and the Shugan-Yishen formula plus TAM group had higher inhibition rates at 48th hour, the inhibition rates was（6.61±0.129）%, （47.43±2.24）%, （54.19±3.364）%, compared to the blank group, Shugan-Yishen formula group and the Shugan-Yishen formula plus TAM group had a stronger inhibition on LCC9 at 48th hour （P〈0.05）, and the combination group was better than Shugan-Yishen formula alone（P〈0.05）, there was no difference between TAM group compared to the blank group（P〉0.05）. RT-PCR result showed that compared to the blank group, the expression levels of ESR1 mRNA of group of Shugan Yishen formula plus TAM＆amp;nbsp;werehigher by and large at 48th hour （P〈0.05）, the expression of ESR1 mRNA was（2.282±0.185）;and the levels of ERBB2, MAPK3 mRNAwere lower（P〈0.05）, which levels were（0.71±0.095）, （0.336±0.058）. Western blotting result showed that Shugan-Yishen Formula plus TAM group could markedly decrease the expression of HER2, p-ERK/MAPK of LCC9 at 48th hour （P〈0.05）, which the expressions were（0.708± 0.072）, （0.228±0.034）, and the expression of ER-αelevated（P〈0.05）. There was no significant difference on ERK/MAPK（P〉0.05）. Conclusions ①The combination of Shugan-Yishen formula with tamoxifen can inhibit the LCC9 cells. The inhibition effect reaches the peak at 48th hour. ②The mechanism of Shugan-Yishen formula plus TAM combination effects on human breast cancer TAM-resistant cell line LCC9 may improve the sensitivity of ERαby inhibiting the expression of HER2, ERK/MAPK. Key words: Shugan-Yishen Formula; Breast cancer; HER2-ERK/MAPK-ERαpathway

Aromatase induction in tamoxifen-resistant breast cancer: Role of phosphoinositide 3-kinase-dependent CREB activation

Estrogens are important for the development and growth of estrogen receptor (ER)-positive breast cancer, for which anti-estrogen therapy is one of the most effective treatments. However, its efficacy can be limited by either de novo or acquired resistance. Aromatase is a key enzyme for the biosynthesis of estrogens, and inhibition of this enzyme leads to profound hypoestrogenism. Here, we found that the basal expression and activity of aromatase were significantly increased in tamoxifen (TAM)-resistant human breast cancer (TAMR-MCF-7) cells compared to control MCF-7 cells. We further revealed that aromatase immunoreactivity in tumor tissues was increased in recurrence group after TAM therapy compared to non-recurrence group after TAM therapy. Phosphorylation of Akt, extracellular signal-regulated kinase (ERK), and p38 kinase were all increased in TAMR-MCF-7 cells. Inhibition of phosphoinositide 3-kinase (PI3K) suppressed the transactivation of the aromatase gene and its enzyme activity. Furthermore, we have also shown that PI3K/Akt-dependent cAMP-response element binding protein (CREB) activation was required for the enhanced expression of aromatase in TAMR-MCF-7 cells. Our findings suggest that aromatase expression is up-regulated in TAM-resistant breast cancer via PI3K/Akt-dependent CREB activation.

Tamoxifen-resistant breast cancer cells possess cancer stem-like cell properties

Background Cancer stem cells (CSCs) are the cause of cancer recurrence because they are resistant to conventional therapy and contribute to cancer growth and metastasis. Endocrinotherapy is the most common breast cancer therapy and acquired tamoxifen (TAM) resistance is the main reason for endocrinotherapy failure during such therapy. Although acquired resistance to endocrine treatment has been extensively studied, the underlying mechanisms are unclear. We hypothesized that breast CSCs played an important role in TAM-induced resistance during breast cancer therapy. Therefore, we investigated the biological characteristics of TAM-resistant (TAM-R) breast cancer cells. Methods Mammosphere formation and tumorigenicity of wild-type (WT) and TAM-R MCF7 cells were tested by a mammosphere assay and mouse tumor xenografts respectively. Stem-cell markers (SOX-2, OCT-4, and CD133) and epithelial-mesenchymal transition (EMT) markers were tested by quantitative real-time (qRT)-PCR. Morphological observation was performed to characterize EMT. Results After induction of TAM resistance, TAM-R MCF7 cells exhibited increased proliferation in the presence of TAM compared to that of WT MCF7 cells (P &lt;0.05), indicating enhanced TAM resistance of TAM-R MCF7 cells compared to that of WT MCF7 cells. TAM-R MCF7 cells showed enhanced mammosphere formation and tumorigenicity in nude mice compared to that of WT MCF7 cells (P &lt;0.01), demonstrating the elevated CSC properties of TAM-R MCF7 cells. Consistently, qRT-PCR revealed that TAM-R MCF7 cells expressed increased mRNA levels of stem cell markers including SOX-2, OCT-4, and CD133, compared to those of WT MCF7 cells (P &lt;0.05). Morphologically, TAM-R MCF7 cells showed a fibroblastic phenotype, but WT MCF7 cells were epithelial-like. After induction of TAM resistance, qRT-PCR indicated that MCF7 cells expressed increased mRNA levels of Snail, vimentin, and N-cadherin and decreased levels of E-cadherin, which are considered as EMT characteristics (P &lt;0.05). Conclusion TAM-R MCF7 cells possess CSC characteristics and may be responsible for TAM resistance during breast cancer therapy.

Abstract 3570: Phospho-dependent regulation of ERRγ expression, transcriptional activity, and Tamoxifen resistance in ER+ breast cancer.

Abstract Selective estrogen receptor modulators (SERMs) such as Tamoxifen (TAM) can significantly improve breast cancer-specific survival for women with ER-positive (ER+) disease. However, resistance to TAM remains a major clinical problem. Estrogen-related receptor gamma (ERRγ) is an orphan nuclear receptor with broad, structural similarities to classical ER that is widely implicated in the transcriptional regulation of energy homeostasis. We previously reported that ERRγ is upregulated during the acquisition of TAM resistance in ER+ breast cancer cell lines, exogenous expression of ERRγ is sufficient to induce TAM resistance, and ERRγ mRNA is significantly increased in tumor samples from women with ER+ breast cancer who relapse following TAM treatment. Because ERRγ has no known ligand, our recent studies have focused on understanding how the expression and activity of this orphan nuclear receptor is regulated, and how this contributes to the TAM resistant phenotype. We have found that TAM-resistant breast cancer cells in which endogenous ERRγ is upregulated show a concomitant hyper activation of p44/p42 ERK. Furthermore, ERK activity (but not that of JNK or p38 MAPK) directly enhances ERRγ protein stability via Serines 57, 81, and/or 219 of the receptor. Phospho-deficient ERRγ is impaired in its ability to induce TAM resistance, and this is associated with a significant reduction in transcriptional activity at the estrogen-related response element (ERRE) half-site, in particular. This led us to hypothesize that ERRγ action at ERREs is most relevant to the development of TAM resistance. We examined a meta-list of validated, ERRE-containing ERR target genes in association with distant metastasis-free survival (DMFS) within 5 years of primary diagnosis in each of 3 publicly available clinical datasets comprised of ER+ breast cancer patients treated with TAM, and obtained a list of 37 differentially expressed targets. The proximal promoter regions of these 37 genes are enriched for binding sites for ELK1, a well-known ERK substrate. Sub-network analysis reveals enrichment of genes whose protein products regulate the mitochondrial unfolded protein response (UPRmito). We also performed differential dependency network (DDN) analysis in an independent dataset using the full meta-list of ERRγ target genes and also identified genes associated with UPRmito. These data suggest a potentially novel role for ERK-mediated regulation of ERRγ in mitochondrial protein folding in TAM-resistant breast cancer. Citation Format: Rebecca B. Riggins, Mary M. Heckler, Hemang Thakor, Cara C. Schafer, Salendra Singh, Ye Tian, Yuriy Gusev, Subha Madhavan, Yue Wang. Phospho-dependent regulation of ERRγ expression, transcriptional activity, and Tamoxifen resistance in ER+ breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3570. doi:10.1158/1538-7445.AM2013-3570

Dose-dependent effect of tamoxifen in tamoxifen-resistant breast cancer cells via stimulation by the ERK1/2 and AKT signaling pathways

The majority of breast cancers undergo progression from an initially endocrine responsive phenotype to an endocrine therapy-resistant phenotype, and acquired resistance to tamoxifen (Tam) is a major clinical problem. In the present study, we aimed to identify the function and mechanism of Tam at different concentrations in cells with acquired Tam resistance. Estrogen-dependent MCF-7 cells were cultured with Tam to generate Tam-resistant (TAM-R) breast cancer cells or in estrogen-free medium to mimic the effects of clinical treatment. In addition, we analyzed the effects of different concentrations of Tam on TAM-R cells by cell counting. Furthermore, the crosstalk between the stimulatory G protein α subunit (Gαs) and the activation of ERK1/2 and AKT in TAM-R cells was examined by small interfering RNA (siRNA) and immunoblotting methods. Low-dose Tam was found to act as an estrogen agonist via stimulation of the ERK1/2 signaling pathway, resulting in acquired resistance to Tam, whereas high-dose Tam inhibited TAM-R cell growth by blocking the activation of ERK1/2 and AKT. Moreover, Gαs was involved in Tam resistance in breast cancer cells. Taken together, our study demonstrated a dose-dependent growth response to Tam in TAM-R cells, which will promote the understanding of the importance of the appropriate use and dosage of Tam in the clinic.

Amurensin G inhibits angiogenesis and tumor growth of tamoxifen-resistant breast cancer via Pin1 inhibition

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem among estrogen-receptor-positive breast cancer patients. We have previously reported that TAM-resistant MCF-7 (TAMR-MCF-7) cells have elevated angiogenic potential via Pin1-dependent vascular endothelial growth factor (VEGF) production. Vitis amurensis grape consumed as wine and fruit contains several resveratrol-like stilbenes or oligostilbenes. In this study, we screened for the most active compound to inhibit VEGF production from V. amurensis. Among the tested compounds, amurensin G most potently suppressed VEGF production in TAMR-MCF-7 cells. The enhanced VEGF gene transcription in TAMR-MCF-7 cells was suppressed by amurensin G. Molecular analyses using reporter genes with hypoxia response elements and activator protein-1 (AP-1) elements, and western blots revealed that the activities and the nuclear levels of hypoxia inducible factor-1 (HIF-1)α and AP-1 in TAMR-MCF-7 cells were decreased by amurensin G. Moreover, amurensin G concentration-dependently inhibited protein expression and gene transcription of Pin1 in TAMR-MCF-7 cells, which was dependent on E2F1 inhibition. Chick chorioallantoic membrane assays confirmed that amurensin G had significant antiangiogenic and antitumor growth effects in TMAR-MCF-7 cells. These results demonstrate for the first time that amurensin G may have therapeutic potential for TAM-resistant breast cancer through blocking of Pin1-mediated VEGF gene transcription.

Differential gene expression in tamoxifen-resistant breast cancer cells revealed by a new analytical model of RNA-Seq data.

Resistance to tamoxifen (Tam), a widely used antagonist of the estrogen receptor (ER), is a common obstacle to successful breast cancer treatment. While adjuvant therapy with Tam has been shown to significantly decrease the rate of disease recurrence and mortality, recurrent disease occurs in one third of patients treated with Tam within 5 years of therapy. A better understanding of gene expression alterations associated with Tam resistance will facilitate circumventing this problem. Using a next generation sequencing approach and a new bioinformatics model, we compared the transcriptomes of Tam-sensitive and Tam-resistant breast cancer cells for identification of genes involved in the development of Tam resistance. We identified differential expression of 1215 mRNA and 513 small RNA transcripts clustered into ERα functions, cell cycle regulation, transcription/translation, and mitochondrial dysfunction. The extent of alterations found at multiple levels of gene regulation highlights the ability of the Tam-resistant cells to modulate global gene expression. Alterations of small nucleolar RNA, oxidative phosphorylation, and proliferation processes in Tam-resistant cells present areas for diagnostic and therapeutic tool development for combating resistance to this anti-estrogen agent.

Abstract 1096: Loss of miR-200 family of microRNAs confers resistance to tamoxifen in human breast cancer cells

Abstract MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level. MiR-200 family includes two clusters i.e. miR-200 a/200b/ 429 and miR-200c/141 encoded on chromosome 1 and chromosome 12, respectively. Previous studies have demonstrated that miR-200 family targets ZEB1, a transcription factor that promotes epithelial-to-mesenchymal transition (EMT). Previous reports demonstrated a loss of miR-200a, b and c in MDA-MB-231 breast cancer cells resulted in EMT. These studies also showed a concomitant increase in ZEB1 protein. We observed lower miR-200a, b and c expression in estrogen-independent LCC1 and endocrine-resistant LCC2, LCC9, and LY2 compared to the parental, endocrine-sensitive MCF-7 human breast cancer cell line. We found that ZEB1 protein is expressed in tamoxifen-resistant LY2 cells but not in tamoxifen-sensitive MCF-7 cells. LY2 cells did not express E-cadherin, a ZEB1 target which is also a marker for epithelial phenotype. This is the first demonstration that LY2 cells have undergone EMT as part of their endocrine-resistant phenotype. These studies indicate that loss of miR-200 and a corresponding increase in ZEB1 protein is an indicator of tamoxifen-resistance in breast cancer cells. Further, in concordance with higher miR-221/222, there was decreased expression of estrogen receptor alpha (ERα) mRNA and protein in LY2 cells when compared to MCF-7 cells. We hypothesize that ERα expression is decreased in cells that have undergone EMT and this subsequently leads to endocrine-resistance and an invasive phenotype. Future studies will determine whether re-expression of ERα or knockdown of ZEB1 restores TAM-sensitivity in LY2 cells. By evaluating the expression of E-cadherin, it will be possible to determine whether restoration of ERα or knockdown of ZEB1 can revert cells from a mesenchymal to an epithelial phenotype. If validated, loss of miR-200 could be used as a marker of poor prognosis in TAM-resistant breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1096. doi:1538-7445.AM2012-1096

A new synthetic HDAC inhibitor, MHY218, induces apoptosis or autophagy-related cell death in tamoxifen-resistant MCF-7 breast cancer cells

Acquired resistance to tamoxifen (Tam) is a critical problem in breast cancer therapy. Therefore, new potential strategies for Tam-resistant breast cancer are needed recently. In this study, we synthesized a novel histone deacetylase (HDAC) inhibitor, MHY218, for the development of potent inhibitors of HDAC and evaluated its biological activities by monitoring the anticancer effects in Tam-resistant MCF-7 (TAMR/MCF-7) cells via in vitro and in vivo studies. MHY218 significantly inhibited the proliferation of TAMR/MCF-7 cells in a dose-dependent manner. The total HDAC enzyme activity was significantly inhibited, corresponding with inhibition of acetylated H3 and H4 expression in TAMR/MCF-7 cells. HDAC1, 4, and 6 expression levels were decreased in response to MHY218 treatment. Cell cycle analysis indicated that MHY218 induced G2/M phase cell cycle arrest. As expected, apoptotic cell death was observed in response to MHY218 treatment. Interestingly, levels of beclin-1 and LC3-II, the markers of autophagy, were increased in TAMR/MCF-7 cells treated with MHY218. The efficacy of MHY218 was also compared with that of SAHA in vivo in a xenograft model of nude mice bearing a TAMR/MCF-7 cells. MHY218 (10 mg/kg, twice a week for 21 days) completely inhibited tumor growth and MHY218 markedly inhibited the expression of proliferative cell nuclear antigen (PCNA) in tumor tissue. These results indicate that MHY218 can induce caspase-independent autophagic cell death rather than apoptotic cell death. The MHY218-induced autophagic cell death could be a new strategy in the treatment of Tam-resistant human breast cancer.