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The clinical studies for breast cancer (BC) are now assessing the efficacy of 2-Methoxyestradiol (2-ME), a naturally occurring derivative of estradiol. Our study aimed to explore the potential of combining the 2-ME and tamoxifen (TAM) on sensitization of TAM-resistant cells using LCC2 the TAM-resistant cells as a model and comparing the results to the sensitive cells MCF-7. Sulphorhodamine-B (SRB) assay is used to examine the 2-ME chemo-sensitizing impact on the cytotoxicity of TAM on LCC2 cells. Colorimetric assay kits were used to assess the level of the apoptosis-related markers caspases 3, Bcl2, and Bax in cell lysate. Hypoxia-inducible factor 1 alpha (HIF-1α) expression was measured using western blotting. Total cholesterol and triglyceride (TG) levels were examined colorimetrically, using the BIOLABO kit. The use of 2-ME enhanced the cytotoxic effects of TAM and effectively reversed TAM resistance. This was achieved by inhibiting the expression of HIF-1α, while concurrently increasing the levels of apoptotic marker caspase-3, as well as the pro-apoptotic protein Bax. Additionally, there was a reduction in the levels of Bcl2, an anti-apoptotic protein. Furthermore, a reduction in TG and cholesterol levels was noted. Our findings show that HIF-1α plays an important role in TAM resistance and that suppression of HIF-1α by 2-ME-mediated sensitization of BC-resistant cells to TAM. Therefore, the concurrent administration of TAM/2-ME might potentially serve as a viable therapeutic approach to address TAM resistance and enhance the overall therapy efficacy for patients with BC. [ABSTRACT FROM AUTHOR]

Estrogen receptor positive (ER+) breast cancer (BCa) accounts for the highest proportion of breast cancer-related deaths. While endocrine therapy is highly effective for this subpopulation, endocrine resistance remains a major challenge and the identification of novel targets is urgently needed. Previously, we have shown that Semaphorin 3C (SEMA3C) is an autocrine growth factor that drives the growth and treatment resistance of various cancers, but its role in breast cancer progression and endocrine resistance is poorly understood. Here, we report that SEMA3C plays a role in maintaining the growth of ER+ BCa cells and is a novel, tractable therapeutic target for the treatment of ER+ BCa patients. Analyses of publicly available clinical datasets indicate that ER+ BCa patients express significantly higher levels of SEMA3C mRNA than other subtypes. Furthermore, SEMA3C mRNA expression was positively correlated with ESR1 mRNA expression. ER+ BCa cell lines (MCF7 and T47D) expressed higher levels of SEMA3C mRNA and protein than a normal mammary epithelial MCF10A cell line. ER siRNA knockdown was suppressed, while dose-dependent beta-estradiol treatment induced SEMA3C expression in both MCF7 and T47D cells, suggesting that SEMA3C is an ER-regulated gene. The stimulation of ER+ BCa cells with recombinant SEMA3C activated MAPK and AKT signaling in a dose-dependent manner. Conversely, SEMA3C silencing inhibited Estrogen Receptor (ER) expression, MAPK and AKT signaling pathways while simultaneously inducing apoptosis, as monitored by flow cytometry and Western blot analyses. SEMA3C silencing significantly inhibited the growth of ER+ BCa cells, implicating a growth dependency of ER+ BCa cells on SEMA3C. Moreover, the analysis of tamoxifen resistant (TamR) cell models (TamC3 and TamR3) showed that SEMA3C levels remain high despite treatment with tamoxifen. Tamoxifen-resistant cells remained dependent on SEMA3C for growth and survival. Treatment with B1SP Fc fusion protein, a SEMA3C pathway inhibitor, attenuated SEMA3C-induced signaling and growth across a panel of tamoxifen sensitive and resistant ER+ breast cancer cells. Furthermore, SEMA3C silencing and B1SP treatment were associated with decreased EGFR signaling in TamR cells. Here, our study implicates SEMA3C in a functional role in ER+ breast cancer signaling and growth that suggests ER+ BCa patients may benefit from SEMA3C-targeted therapy. [ABSTRACT FROM AUTHOR]

Abstract Background Tamoxifen resistance presents a huge clinical challenge for breast cancer patients. An understanding of the mechanisms of tamoxifen resistance can guide development of efficient therapies to prevent drug resistance. Methods We first tested whether peptidylarginine deiminase 2 (PAD2) may be involved in tamoxifen-resistance in breast cancer cells. The effect of depleting or inhibiting PAD2 in tamoxifen-resistant MCF-7 (MCF7/TamR) cells was evaluated both in vitro and in vivo. We then investigated the potential of Cl-amidine, a PAD inhibitor, to be used in combination with tamoxifen or docetaxel, and further explored the mechanism of the synergistic and effective drug regimen of PADs inhibitor and docetaxel on tamoxifen-resistant breast cancer cells. Results We report that PAD2 is dramatically upregulated in tamoxifen-resistant breast cancer. Depletion of PAD2 in MCF7/TamR cells facilitated the sensitivity of MCF7/TamR cells to tamoxifen. Moreover, miRNA-125b-5p negatively regulated PAD2 expression in MCF7/TamR cells, therefore overexpression of miR-125b-5p also increased the cell sensitivity to tamoxifen. Furthermore, inhibiting PAD2 with Cl-amidine not only partially restored the sensitivity of MCF7/TamR cells to tamoxifen, but also more efficiently enhanced the efficacy of docetaxel on MCF7/TamR cells with lower doses of Cl-amidine and docetaxel both in vivo and in vivo. We then showed that combination treatment with Cl-amidine and docetaxel enhanced p53 nuclear accumulation, which synergistically induced cell cycle arrest and apoptosis. Meanwhile, p53 activation in the combination treatment also accelerated autophagy processes by synergistically decreasing the activation of Akt/mTOR signaling, thus enhancing the inhibition of proliferation. Conclusion Our results suggest that PAD2 functions as an important new biomarker for tamoxifen-resistant breast cancers and that inhibiting PAD2 combined with docetaxel may offer a new approach to treatment of tamoxifen-resistant breast cancers.

Tamoxifen (TAM) is a key player in estrogen receptor-positive (ER+) breast cancer (BC); however, ∼30% of patients experience relapse and a lower survival rate due to TAM resistance. TAM resistance was related to the over expression of SOX-2 gene, which is regulated by the E2F3 transcription factor in the Wnt signaling pathway. It was suggested that SOX-2 overexpression was suppressed by dexamethasone (DEX), a glucocorticoid commonly prescribed to BC patients. The aim of the present study is to explore the effect of combining DEX and TAM on the inhibition of TAM-resistant LCC-2 cells (TAMR-1) through modulating the E2F3/SOX-2-mediated Wnt signaling pathway. The effect of the combination therapy on MCF-7 and TAMR-1 cell viability was assessed. Drug interactions were analyzed using CompuSyn and SynergyFinder softwares. Cell cycle distribution, apoptotic protein expression, gene expression levels of SOX-2 and E2F3, and cell migration were also assessed. Combining DEX with TAM led to synergistic inhibition of TAMR-1 cell proliferation and migration, induced apoptosis, reduced SOX-2 and E2F3 expression and was also associated with S and G2-M phase arrest. Therefore, combining DEX with TAM may present an effective therapeutic option to overcome TAM resistance, by targeting the E2F3/SOX-2/Wnt signaling pathway, in addition to its anti-inflammatory effect., (© 2024 The Author(s).)

Background: Breast cancer is still one of the major public health burdens worldwide, although there is tremendous progress in early diagnosis and treatment of breast cancer. Tamoxifen was one of the most popular endocrine therapies for early-stage estrogen receptor (ER) + breast cancer patients. However, a high incidence of drug resistance develops along with poor prognosis in breast cancer. Currently, long noncoding RNAs (lncRNAs) are emerging and are well suited to play a role in the development of cancer and tamoxifen resistance. However, there is little reported about the relationship of breast cancer resistance to tamoxifen and lncRNA H19. Here, we validated that lncRNA H19 was highly expressed in breast cancer especially in patients with late stage (III and IV), compared to normal tissues and early stage cancers (I and II).Methods: Quantitative polymerase chain reaction (qPCR) was utilized for comparison of lncRNA H19 expression level in breast cancers with different stages. qPCR and Western blotting were used to detect gene and protein, respectively.Results: We found that lncRNA H19 expression level manipulated breast cancer cell proliferation both in parental breast cancer cell lines and tamoxifen-resistant cell lines. Knockdown of lncRNA H19 elevated tamoxifen sensitivity for promoting cell growth and inhibiting apoptosis in tamoxifen-resistant breast cancer cells. Moreover, knockdown of H19 inhibited Wnt pathway and epithelia–mesenchymal transition in tamoxifen-resistance breast cancer cells.Conclusion: Taken together, the results of this study provided the evidence for H19 in regulating tamoxifen-resistant breast cancer and might provide novel options in the future treatment of tamoxifen-resistance breast cancer patients. [ABSTRACT FROM AUTHOR]

Endocrine therapy using antiestrogens and aromatase inhibitors is usually efficient to treat patients with hormone-sensitive breast cancer. Many patients with endocrine therapy, however, often acquire resistance. In the present study, we performed functional screening using short hairpin RNA library to dissect genes involved in antiestrogen tamoxifen resistance in MCF-7 breast cancer cells. We identified seven candidate genes that are associated with poor prognosis of breast cancer patients based on clinical dataset. The expression levels of six out of seven genes were higher in 4-hydroxytamoxifen (OHT) resistant MCF-7 (OHTR) cells compared with parental MCF-7 cells. Among the six selected genes, siRNA-mediated knockdown of PSMD1 and TSPAN12 markedly reduced the proliferation of OHTR cells. Notably, the knockdown of proteasome 26S subunit PSMD1 exhibited cell cycle arrest and the accumulation of p53 protein through inhibiting p53 protein degradation. In accordance with p53 accumulation, its target genes p21 and SFN were also upregulated by PSMD1 silencing. Taken together, PSMD1 was identified as a potential gene that plays a role in the development of tamoxifen resistance in breast cancer cells. These findings will provide a new insight for the mechanism underlying endocrine therapy resistance and a prognostic and therapeutic molecular target for advanced breast cancer. [ABSTRACT FROM AUTHOR]

Acquired tamoxifen (TAM) resistance is a significant clinical problem in treating patients with estrogen receptor α (ERα)+ breast cancer. We reported that ERα increases nuclear respiratory factor-1 (NRF-1), which regulates nuclear-encoded mitochondrial gene transcription, in MCF-7 breast cancer cells and NRF-1 knockdown stimulates apoptosis. Whether NRF-1 and target gene expression is altered in endocrine resistant breast cancer cells is unknown. We measured NRF-1and metabolic features in a cell model of progressive TAM-resistance. NRF-1 and its target mitochondrial transcription factor A (TFAM) were higher in TAM-resistant LCC2 and LCC9 cells than TAM-sensitive MCF-7 cells. Using extracellular flux assays we observed that LCC1, LCC2, and LCC9 cells showed similar oxygen consumption rate (OCR), but lower mitochondrial reserve capacity which was correlated with lower Succinate Dehydrogenase Complex, Subunit B in LCC1 and LCC2 cells. Complex III activity was lower in LCC9 than MCF-7 cells. LCC1, LCC2, and LCC9 cells had higher basal extracellular acidification (ECAR), indicating higher aerobic glycolysis, relative to MCF-7 cells. Mitochondrial bioenergetic responses to estradiol and 4-hydroxytamoxifen were reduced in the endocrine-resistant cells compared to MCF-7 cells. These results suggest the acquisition of altered metabolic phenotypes in response to long term antiestrogen treatment may increase vulnerability to metabolic stress. [ABSTRACT FROM AUTHOR]

Our previous studies demonstrated that specific inhibition of the BIG3- PHB2 complex, which is a critical modulator in estrogen (E2) signaling, using ERAP, a dominant negative peptide inhibitor, leads to suppression of E2-dependent estrogen receptor ( ER) alpha activation through the reactivation of the tumor suppressive activity of PHB2. Here, we report that ERAP has significant suppressive effects against synergistic activation caused by the crosstalk between E2 and growth factors associated with intrinsic or acquired resistance to anti-estrogen tamoxifen in breast cancer cells. Intrinsic PHB2 released from BIG3 by ERAP effectively disrupted each interaction of membrane-associated ERα and insulin-like growth factor 1 receptor beta ( IGF-1Rβ), EGFR, PI3K or human epidermal growth factor 2 ( HER2) in the presence of E2 and the growth factors IGF or EGF, followed by inhibited the activation of IGF-1Rβ, EGFR or HER2, and reduced Akt, MAPK and ERα phosphorylation levels, resulting in significant suppression of proliferation of ERα-positive breast cancer cells in vitro and in vivo. More importantly, combined treatment with ERAP and tamoxifen led to a synergistic suppression of signaling that was activated by crosstalk between E2 and growth factors or HER2 amplification. Taken together, our findings suggest that the specific inhibition of BIG3- PHB2 is a novel potential therapeutic approach for the treatment of tamoxifen-resistant breast cancers activated by the crosstalk between E2 and growth factor signaling, especially in premenopausal women. [ABSTRACT FROM AUTHOR]

Highlights: [•] 1α,25-Dihydroxyvitamin D (1,25D) stimulates gene expression of NFκB inhibitor IκB. [•] 1,25D strongly decrease growth of tamoxifen-resistant breast cancer cells. [•] Cells treated with 1,25D have a decreased sensitivity to TNFα stimulation. [•] 1,25D strongly decreases the TNFα-induced translocation of p65 into the nucleus. [ABSTRACT FROM AUTHOR]

Background: Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor known to interact with growth factor receptors, is upregulated in tamoxifen-resistant (TamR) MCF7 breast cancer cells. The purpose of this study was to explore the consequences of CD44 upregulation in an MCF7 cell model of acquired tamoxifen resistance, specifically with respect to the hypothesis that CD44 may influence erbB activity to promote an adverse phenotype. Methods: CD44 expression in MCF7 and TamR cells was assessed by RT-PCR, Western blotting and immunocytochemistry. Immunofluorescence and immunoprecipitation studies revealed CD44-erbB associations. TamR cells (± siRNA-mediated CD44 suppression) or MCF7 cells (± transfection with the CD44 gene) were treated with the CD44 ligand, hyaluronon (HA), or heregulin and their in vitro growth (MTT), migration (Boyden chamber and wound healing) and invasion (Matrigel transwell migration) determined. erbB signalling was assessed using Western blotting. The effect of HA on erbB family dimerisation in TamR cells was determined by immunoprecipitation in the presence or absence of CD44 siRNA. Results: TamR cells overexpressed CD44 where it was seen to associate with erbB2 at the cell surface. siRNA-mediated suppression of CD44 in TamR cells significantly attenuated their response to heregulin, inhibiting heregulin-induced cell migration and invasion. Furthermore, TamR cells exhibited enhanced sensitivity to HA, with HA treatment resulting in modulation of erbB dimerisation, ligand-independent activation of erbB2 and EGFR and induction of cell migration. Overexpression of CD44 in MCF7 cells, which lack endogenous CD44, generated an HA-sensitive phenotype, with HA-stimulation promoting erbB/EGFR activation and migration. Conclusions: These data suggest an important role for CD44 in the context of tamoxifen-resistance where it may augment cellular response to erbB ligands and HA, factors that are reported to be present within the tumour microenvironment in vivo. Thus CD44 may present an important determinant of breast cancer progression in the setting of endocrine resistance. [ABSTRACT FROM AUTHOR]

Abstract: Microarrays identified miRNAs differentially expressed and 4-hydroxytamoxifen (4-OHT) regulated in MCF-7 endocrine-sensitive versus resistant LY2 human breast cancer cells. 97 miRNAs were differentially expressed in MCF-7 versus LY2 cells. Opposite expression of miRs-10a, 21, 22, 29a, 93, 125b, 181, 200a, 200b, 200c, 205, and 222 was confirmed. Bioinformatic analyses to impute the biological significance of these miRNAs identified 36 predicted gene targets from those regulated by 4-OHT in MCF-7 cells. Agreement in the direction of anticipated regulation was detected for 12 putative targets. These miRNAs with opposite expression between the two cell lines may be involved in endocrine resistance. [Copyright &y& Elsevier]

Acquired resistance to endocrine therapies presents a major obstacle to the successful treatment of breast cancer patients. Previously, we have shown that acquisition of resistance to tamoxifen in breast cancer cells is accompanied by an elevation in Src kinase activity which promotes an aggressive, invasive phenotype in vitro. Here, we have explored the potential therapeutic effects of combining Src inhibition with anti-oestrogen treatment on the development of endocrine insensitivity in breast cancer cells. Treatment of MCF7 and T47D cells with tamoxifen alone resulted in an initial growth inhibitory phase followed by the eventual development of tamoxifen resistance together with an elevation of Src kinase activity, which was central to their increased invasive capacity. Chronic exposure of both cell types to the Src inhibitor, AZD0530, as a monotherapy resulted in outgrowth of AZD0530-resistant cells, in which Src kinase activity remained suppressed as did their in vitro invasive nature. Treatment of both MCF7 and T47D cells with AZD0530 in combination with tamoxifen resulted in a reduction of Src activity together with inhibition of focal adhesion kinase phosphorylation and a complete abrogation of their in vitro invasive behaviour. Furthermore, combination therapy significantly suppressed expression of cyclinD1 and c-myc and prevented cell proliferation and the subsequent emergence of a resistant phenotype, with total cell loss occurring by 12 weeks. These data demonstrate that pharmacological targeting of Src kinase, in conjunction with antihormone therapies, effectively prevents antihormone resistance in breast cancer cells in vitro and suggests a potential novel therapeutic benefit of Src kinase inhibitors clinically. [ABSTRACT FROM AUTHOR]

Abstract: Tamoxifen is the drug most used for early breast cancer treatment in oestrogen receptor (ER) positive patients. Unfortunately, despite high ER tumour levels in a tumour, resistance to endocrine therapy, either de novo or acquired after prolonged treatment, can occur. In this review, we will try to summarise the postulated mechanisms of hormonal-resistance, namely, the role of co-regulators and the crosstalk between the HER-2, IGF-IR, Cox-2 and ER pathways. Other predictive markers of tamoxifen-resistance/response, such as cyclin E and UPA/PAI-1, are also discussed. [Copyright &y& Elsevier]

The H19 long non-coding RNA is involved in the development of tamoxifen resistance in breast cancer. However, the relationship between H19 and the metastatic potential and treatment options for tamoxifen-resistant (TAMR) breast cancer is not completely understood. Curcumin inhibits cellular proliferation, migration and invasiveness in several cancer types, including pancreatic cancer, breast cancer and chronic myeloid leukemia. The present study aimed to investigate the role of H19 in MCF-7/TAMR cell epithelial-mesenchymal transition (EMT), migration and invasiveness, and to assess the ability of curcumin to inhibit H19-mediated effects. Reverse transcription-quantitative PCR and western blot analysis were conducted to detect the gene or protein expression. Cell Counting Kit-8, wound healing and Transwell invasion assays were performed to estimate the capabilities of cell viability, invasion and migration. H19 overexpression enhanced MCF-7/TAMR cell EMT, invasion and migration by upregulating Snail. Furthermore, curcumin notably decreased the expression levels of epithelial marker E-cadherin and markedly increased the expression levels of mesenchymal marker N-cadherin in MCF-7/TAMR cells compared with the control group. In addition, following treatment with curcumin for 48 h, H19 expression was decreased in a dose-dependent manner. Moreover, curcumin treatment for 48 h significantly attenuated H19-induced alterations in N-cadherin and E-cadherin expression levels. Curcumin also prevented H19-induced invasion and migration. The present study indicated that H19 may serve as a promoting factor of EMT, invasion and migration in MCF-7/TAMR cells, suggesting that curcumin may prevent H19-associated metastasis. Therefore, curcumin may serve as a promising therapeutic drug for patients with TAMR breast cancer. [ABSTRACT FROM AUTHOR]

The H19 long non‑coding RNA is involved in the development of tamoxifen resistance in breast cancer. However, the relationship between H19 and the metastatic potential and treatment options for tamoxifen‑resistant (TAMR) breast cancer is not completely understood. Curcumin inhibits cellular proliferation, migration and invasiveness in several cancer types, including pancreatic cancer, breast cancer and chronic myeloid leukemia. The present study aimed to investigate the role of H19 in MCF‑7/TAMR cell epithelial‑mesenchymal transition (EMT), migration and invasiveness, and to assess the ability of curcumin to inhibit H19‑mediated effects. Reverse transcription‑quantitative PCR and western blot analysis were conducted to detect the gene or protein expression. Cell Counting Kit‑8, wound healing and Transwell invasion assays were performed to estimate the capabilities of cell viability, invasion and migration. H19 overexpression enhanced MCF‑7/TAMR cell EMT, invasion and migration by upregulating Snail. Furthermore, curcumin notably decreased the expression levels of epithelial marker E‑cadherin and markedly increased the expression levels of mesenchymal marker N‑cadherin in MCF‑7/TAMR cells compared with the control group. In addition, following treatment with curcumin for 48 h, H19 expression was decreased in a dose‑dependent manner. Moreover, curcumin treatment for 48 h significantly attenuated H19‑induced alterations in N‑cadherin and E‑cadherin expression levels. Curcumin also prevented H19‑induced invasion and migration. The present study indicated that H19 may serve as a promoting factor of EMT, invasion and migration in MCF‑7/TAMR cells, suggesting that curcumin may prevent H19‑associated metastasis. Therefore, curcumin may serve as a promising therapeutic drug for patients with TAMR breast cancer.

Background: Tamoxifen resistance presents a huge clinical challenge for breast cancer patients. An understanding of the mechanisms of tamoxifen resistance can guide development of efficient therapies to prevent drug resistance. Methods: We first tested whether peptidylarginine deiminase 2 (PAD2) may be involved in tamoxifen-resistance in breast cancer cells. The effect of depleting or inhibiting PAD2 in tamoxifen-resistant MCF-7 (MCF7/TamR) cells was evaluated both in vitro and in vivo. We then investigated the potential of Cl-amidine, a PAD inhibitor, to be used in combination with tamoxifen or docetaxel, and further explored the mechanism of the synergistic and effective drug regimen of PADs inhibitor and docetaxel on tamoxifen-resistant breast cancer cells. Results: We report that PAD2 is dramatically upregulated in tamoxifen-resistant breast cancer. Depletion of PAD2 in MCF7/TamR cells facilitated the sensitivity of MCF7/TamR cells to tamoxifen. Moreover, miRNA-125b-5p negatively regulated PAD2 expression in MCF7/TamR cells, therefore overexpression of miR-125b-5p also increased the cell sensitivity to tamoxifen. Furthermore, inhibiting PAD2 with Cl-amidine not only partially restored the sensitivity of MCF7/TamR cells to tamoxifen, but also more efficiently enhanced the efficacy of docetaxel on MCF7/TamR cells with lower doses of Cl-amidine and docetaxel both in vivo and in vivo. We then showed that combination treatment with Cl-amidine and docetaxel enhanced p53 nuclear accumulation, which synergistically induced cell cycle arrest and apoptosis. Meanwhile, p53 activation in the combination treatment also accelerated autophagy processes by synergistically decreasing the activation of Akt/mTOR signaling, thus enhancing the inhibition of proliferation. Conclusion: Our results suggest that PAD2 functions as an important new biomarker for tamoxifen-resistant breast cancers and that inhibiting PAD2 combined with docetaxel may offer a new approach to treatment of tamoxifen-resistant breast cancers. [ABSTRACT FROM AUTHOR]

HOXB7 is a homeodomain containing transcription factor which plays a pivotal role in tamoxifen resistant breast cancer. Our work has shown that overexpression of HOXB7 renders cells tamoxifen resistant by mobilizing a number of receptor tyrosine kinase pathways. EGFR expression is upregulated by direct binding of HOXB7 to the EGFR promoter, while HOXB7 functions as a cofactor with ERα to cause overexpression of multiple ER-target genes, including HER2, in tamoxifen resistant breast cancer cells. Probing the pathway further, we found that miR-196a and MYC are upstream regulators of HOXB7 expression. Mechanistically, HOXB7 and ERα jointly upregulate HER2 which phosphorylates MYC. Thus stabilized, MYC in turn suppresses miR-196a. Loss of miR-196a results lifts the quelling influence of miR-196a on HOXB7 expression. Besides shedding light on the intricate interplay of events occurring in tamoxifen resistant breast cancer, the work identifies a number of new therapeutic targets capable of restoring sensitivity of breast cancer cells to tamoxifen.