[Rich66]

Int J Oncol. 2007 Feb;30(2):509-20.
Role for HER2/neu and HER3 in fulvestrant-resistant breast cancer.

Osipo C, Meeke K, Cheng D, Weichel A, Bertucci A, Liu H, Jordan VC.
Department of Pathology, Oncology Institute, Cardinal Bernadin Cancer Center, Loyola University Medical Center, Maywood, IL, USA.
Tamoxifen resistance is common for estrogen receptor alpha (ERalpha) positive breast cancer. Second-line therapies include aromatase inhibitors or fulvestrant. We have shown previously that fulvestrant reversed 17beta-estradiol-induced tumor regression of tamoxifen-stimulated MCF-7 xenografts (MCF-7TAMLT) treated for >5 years with tamoxifen in athymic mice and paradoxically stimulated growth. We investigated mechanisms responsible for growth by fulvestrant in the presence of physiologic estradiol and therapeutic strategies in vivo. The results demonstrated that only estradiol increased expression of the estrogen-responsive genes, c-myc, igf-1, cathepsin D, and pS2 mRNAs, in MCF-7E2 and MCF-7TAMLT tumors. Tamoxifen or fulvestrant decreased the estradiol-induced increase of these mRNAs in both tumor models. However, tyrosine-phosphorylated HER2/ neu, HER3, phospho-extracellular-regulated kinase-1/2 (ERK-1/2), and phospho-glycogen synthetase kinase 3alpha (GSK3alpha) and beta proteins were increased in MCF-7TAMLT tumors treated with fulvestrant compared to estradiol, control, or tamoxifen. Phospho-HER2/neu interacted with HER3 protein in MCF-7TAMLT tumors. In order to determine whether the functional interaction of HER2/neu with HER3 is critical for growth of fulvestrant-stimulated MCF-7TAMLT tumors, pertuzumab (an antibody that blocks HER2/neu-HER3 interaction) was used in an in vivo xenograft growth assay. Only growth of fulvestrant-treated MCF-7TAMLT xenografts was decreased significantly by 37.2% in response to pertuzumab (P=0.004). Pertuzumab specifically decreased the interaction of HER2/neu protein with HER3 in fulvestrant-stimulated MCF-7TAMLT tumors. These results suggested growth of MCF-7TAMLT tumors by tamoxifen or fulvestrant is potentially independent of ERalpha transcriptional activity as evidenced by lack of induction of four estrogen-responsive genes. The results suggested that growth of MCF-7TAMLT tumors treated with fulvestrant in the presence of physiologic estradiol is in part mediated through enhanced signaling from the HER2/neu-HER3 pathway as pertuzumab partially inhibited growth and the interaction of HER2/neu with HER3 in vivo.

PMID: 17203234 [PubMed - indexed for MEDLINE]




J Natl Cancer Inst. 2004 Jun 16;96(12):926-35.
Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer.

Shou J, Massarweh S, Osborne CK, Wakeling AE, Ali S, Weiss H, Schiff R.
The Breast Center, Baylor College of Medicine, Houston, TX 77030, USA.
Comment in:

• J Natl Cancer Inst. 2004 Jun 16;96(12):895-7.

BACKGROUND: Patients receiving adjuvant tamoxifen whose tumors express high levels of both HER2/neu (HER2) and the estrogen receptor (ER) coactivator AIB1 often develop tamoxifen resistance. We used a breast cancer model system with high expression of AIB1 and HER2 to investigate the possible mechanisms underlying this resistance. METHODS: MCF-7 breast cancer cells, which express high levels of AIB1, and a tamoxifen-resistant derivative cell line engineered to overexpress HER2 (MCF-7/HER2-18) were treated with estrogen, tamoxifen, epidermal growth factor (EGF), or heregulin in the absence or presence of the EGF receptor (EGFR) tyrosine kinase inhibitor gefitinib. We analyzed phosphorylation of signaling intermediates by immunoblotting, ER transcriptional activity with reporter gene constructs and immunoblot analysis of endogenous gene products, promoter assembly by chromatin immunoprecipitation (ChIP) assay, and tumor cell growth in vitro by anchorage-independent colony formation and in vivo using xenografts in nude mice. RESULTS: MCF-7/HER2-18 tumors were completely growth inhibited by estrogen deprivation but were growth stimulated by tamoxifen. Molecular cross-talk between the ER and HER2 pathways was increased in the MCF-7/HER-2 cells compared with MCF-7 cells, with cross-phosphorylation and activation of both the ER and the EGFR/HER2 receptors, the signaling molecules AKT and ERK 1,2 mitogen-activated protein kinase (MAPK), and AIB1 itself with both estrogen and tamoxifen treatment. Tamoxifen recruited coactivator complexes (ER, AIB1, CBP, p300) to the ER-regulated pS2 gene promoter in MCF-7/HER2-18 cells and corepressor complexes (NCoR, histone deacetylase 3) in MCF-7 cells. Gefitinib pretreatment blocked receptor cross-talk, reestablished corepressor complexes with tamoxifen-bound ER on target gene promoters, eliminated tamoxifen's agonist effects, and restored its antitumor activity both in vitro and in vivo in MCF-7/HER2-18 cells. CONCLUSIONS: Tamoxifen behaves as an estrogen agonist in breast cancer cells that express high levels of AIB1 and HER2, resulting in de novo resistance. Gefitinib's ability to eliminate this cross-talk and to restore tamoxifen's antitumor effects should be tested in the clinic.

PMID: 15199112 [PubMed - indexed for MEDLINE]


Breast Cancer Res Treat. 2005 Aug;92(3):251-63.
In vitro and in vivo effects of combination of Trastuzumab (Herceptin) and Tamoxifen in breast cancer.

Wang CX, Koay DC, Edwards A, Lu Z, Mor G, Ocal IT, Digiovanna MP.
Department of Internal Medicine (Section of Medical Oncology), Yale University School of Medicine, 333 Cedar Street, Room NSB288, 06510, New Haven, CT 06510, USA.
Extensive interactions between estrogen receptor alpha (ERalpha) and HER2 signaling pathways have been described. Using BT-474 human breast cancer cells, we have previously shown that the combination of tamoxifen (TAM) and Herceptin results in strong synergistic growth inhibition, enhancement of G(0)-G(1) cell cycle accumulation, inhibition of HER2 activity and a cytostatic effect without cell death. To further examine the underlying mechanism of synergy, we investigated the effect of this drug combination on ERalpha function and growth factor downstream signaling. TAM caused a small increase in ERalpha levels while Herceptin had no effect, and both drugs caused an increase in the level of Ser118-phosphorylated ERalpha. However, both TAM and Herceptin individually inhibited ERalpha transcriptional activity, although the combination did not have a greater effect than either single agent. Herceptin inhibited MAPK and Akt activity, while TAM had no effect on these either as a single agent or when added to Herceptin. Using a BALB/c athymic BT-474 in vivo xenograft model, the drug combination (Herceptin 0.3 mg/kg i.p. twice weekly, TAM 1.0 mg/mouse i.p. three times per week) showed a greater inhibition of tumor growth compared to either single agent. Tumor extracts and fixed sections were examined at the end of the treatment period for treatment-specific alterations: we noted a paradoxical proliferation-inducing effect of TAM that was reversed by the addition of Herceptin. Our results indicate that combined targeting of both peptide growth factor receptors and ERalpha represents a promising breast cancer treatment strategy.




J Steroid Biochem Mol Biol. 2005 Feb;93(2-5):249-56.
Reversal of tamoxifen resistant breast cancer by low dose estrogen therapy.

Osipo C, Gajdos C, Cheng D, Jordan VC.
Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Currently, the standard of care for estrogen receptor (ER)-positive breast cancer is 5 years of tamoxifen (TAM) or an aromatase inhibitor (AI) such as anastrozole. New studies indicate that extending antiestrogen therapy beyond 5 years with sequential regimens will improve disease-free survival. Based on the emerging concept that longer therapies are better, we have developed sequential models of tamoxifen-resistant breast cancer in vivo to mimic the clinical scenario of long-term antiestrogen therapy. The goal of the current study was to investigate the consequences of long-term treatment with tamoxifen on the growth of breast tumors in athymic mice. The results demonstrate that there are distinct phases of resistance to tamoxifen that correlate with time of treatment and expression of HER2/neu mRNA. In the treatment phase, 17beta-estradiol (E2) stimulated growth, while TAM inhibited growth of MCF-7 tumors (MCF-7E2). The withdrawal of treatment, mimicking the use of an AI, completely prevented growth. In Phase I resistance, the tumors (MCF-7TAMST) were growth-stimulated by either E2 or TAM, but inhibited by no treatment, fulvestrant, or E2 + fulvestrant. Phase II-resistant tumors (MCF-7TAMLT) were treated for more than 5 years and growth-stimulated by TAM. However, no treatment, fulvestrant, or E2 completely inhibited growth. Interestingly, the few tumors (MCF-7TAMLT) that survived in response to E2 were robustly re-stimulated by E2 after transplantation into new generations of athymic mice. These E2-stimulated tumors (MCF-7TAME) were inhibited by TAM in a dose-dependent similar to their parental tumors (MCF-7E2). In addition, the MCF-7TAME tumors were inhibited by either no treatment or fulvestrant. HER2/neu and HER3 mRNAs were over-expressed in TAM-stimulated MCF-7TAMLT tumors and remained high in E2-stimulated MCF-7TAME tumors. The data indicate that complete reversal of resistance to TAM can be achieved with the use of low dose E2 therapy. Also, these data suggest that over-expression of HER2/neu alone is insufficient to predict resistance to TAM. Based on the results, we suggest using an alternating treatment regimen, cycling antiestrogen with estrogen therapy to avoid drug-resistance.



PMID: 15860267 [PubMed - indexed for MEDLINE]

Clin Cancer Res. 2007 Aug 15;13(16):4882-90.
Hydroxamic acid analogue histone deacetylase inhibitors (inc Vorinostat) attenuate estrogen receptor-alpha levels and transcriptional activity: a result of hyperacetylation and inhibition of chaperone function of heat shock protein 90.

Fiskus W, Ren Y, Mohapatra A, Bali P, Mandawat A, Rao R, Herger B, Yang Y, Atadja P, Wu J, Bhalla K.
Medical College of Georgia Cancer Center, Augusta, Georgia 30912, USA.
PURPOSE: The molecular chaperone heat shock protein (hsp)-90 maintains estrogen receptor (ER)-alpha in an active conformation, allowing it to bind 17beta-estradiol (E2) and transactivate genes, including progesterone receptor (PR)-beta and the class IIB histone deacetylase HDAC6. By inhibiting HDAC6, the hydroxamic acid analogue pan-HDAC inhibitors (HA-HDI; e.g., LAQ824, LBH589, and vorinostat) induce hyperacetylation of the HDAC6 substrates alpha-tubulin and hsp90. Hyperacetylation of hsp90 inhibits its chaperone function, thereby depleting hsp90 client proteins. Here, we determined the effect of HA-HDIs on the levels and activity of ERalpha, as well as on the survival of ERalpha-expressing, estrogen-responsive human breast cancer MCF-7 and BT-474 cells. EXPERIMENTAL DESIGN: Following exposure to HA-HDIs, hsp90 binding, polyubiquitylation levels, and transcriptional activity of ERalpha, as well as apoptosis and loss of survival, were determined in MCF-7 and BT-474 cells. RESULTS: Treatment with HA-HDI induced hsp90 hyperacetylation, decreased its binding to ERalpha, and increased polyubiquitylation and depletion of ERalpha levels. HA-HDI treatment abrogated E2-induced estrogen response element-luciferase expression and attenuated PRbeta and HDAC6 levels. Exposure to HA-HDI also depleted p-Akt, Akt, c-Raf, and phospho-extracellular signal-regulated kinase-1/2 levels, inhibited growth, and sensitized ERalpha-positive breast cancer cells to tamoxifen. CONCLUSIONS: These findings show that treatment with HA-HDI abrogates ERalpha levels and activity and could sensitize ERalpha-positive breast cancers to E2 depletion or ERalpha antagonists.

PMID: 17699868 [PubMed - indexed for MEDLINE]

1: Mol Cell Endocrinol. 2009 Sep 20. [Epub ahead of print] Links

Valproic acid restores ERalpha and antiestrogen sensitivity to ERalpha-negative breast cancer cells.

Fortunati N, Bertino S, Costantino L, De Bortoli M, Compagnone A, Bandino A, Catalano MG, Boccuzzi G.
Oncological Endocrinology, AUO San Giovanni Battista, Torino, Italy.
Histone deacetylase inhibitors (HDIs) are valuable drugs in breast cancer where estrogen receptor alpha (ERalpha) can be silenced by epigenetic modifications. We report the effect of the clinically available HDI, valproic acid (VPA), on ERalpha expression and function in ER-negative breast cancer cells, MDA-MB-231. VPA induced ERalpha mRNA and protein, while did not modify ERbeta. In VPA-treated cells, we also observed: (1) a correct transcriptional response to estradiol after transfection with the luciferase gene under the control of an estrogen-responsive minimal promoter (ERE-TKluc); (2) increased expression of the ER-related transcription factor FoxA1; (3) estradiol-induced up-regulation of several estrogen-regulated genes (e.g. pS2, progesterone receptor); (4) inhibitory effect of tamoxifen on cell growth. In conclusion, the HDI VPA, inducing ERalpha and FoxA1, confers to MDA-MB 231 cells an estrogen-sensitive "phenotype", restoring their sensitivity to antiestrogen therapy.
PMID: 19772891 [PubMed - as supplied by publisher

Cancer Prev Res (Phila Pa). 2010 Feb 23. [Epub ahead of print]
The Impact of Fish Oil on the Chemopreventive Efficacy of Tamoxifen against Development of N-Methyl-N-Nitrosourea-Induced Rat Mammary Carcinogenesis.

Manni A, Xu H, Washington S, Aliaga C, Cooper T, Richie JP Jr, Bruggeman R, Prokopczyk B, Calcagnotto A, Trushin N, Mauger D, Verderame MF, El-Bayoumy K.
Authors' Affiliations: Departments of 1Medicine, 2Biochemistry and Molecular Biology, 3Comparative Medicine, 4Public Health Sciences, 5Pathology, and 6Pharmacology, Pennsylvania State University College of Medicine, Hershey Medical Center, Hershey, Pennsylvania.
The antiestrogen tamoxifen reduces breast cancer incidence in high-risk women but is unable to inhibit the development of hormone-independent tumors. Omega-3 polyunsaturated fatty acids (n-3 PUFA), known ligands of the peroxisome proliferator activated receptor-gamma (PPARgamma), generally exert tumor-suppressive effects. Based on the known crosstalk between the estrogen and the PPARgamma receptors, we tested the hypothesis that the combination of tamoxifen with n-3 PUFA results in a superior antitumor action over the individual interventions. In this study, we report for the first time that the combination of a fish oil diet rich in n-3 PUFA and tamoxifen seemed to inhibit N-methyl-N-nitrosourea-induced mammary carcinogenesis, tumor multiplicity, and volume to a greater extent than the individual interventions. The potential superiority of the combination was particularly evident at a suboptimal dose of tamoxifen, which, by itself, was unable to significantly decrease tumor development. Because activation of PPARgamma is known to inhibit oxidative stress, we examined the effects of our interventions on circulating and tumor levels of glutathione, a major intracellular antioxidant. Our results indicate that reduction in the level of oxidative stress may be a potential mechanism by which the n-3 PUFA-rich diet potentiated the tumor-suppressive effect of tamoxifen. Our interventions were well tolerated without evidence of toxicity. Combined administration of tamoxifen and n-3 PUFA is a promising new approach to breast cancer prevention. Because of its safety, this combination can quickly be translated to the clinic if its superiority can be supported by future studies. Cancer Prev Res; 3(3); 322-30.

PMID: 20179301 [PubMed - as supplied by publisher]






J Mammary Gland Biol Neoplasia. 2009 Mar;14(1):45-54. Epub 2009 Feb 28.
Resistance to endocrine therapy: are breast cancer stem cells the culprits?
(Full text PDF attached)
O'Brien CS, Howell SJ, Farnie G, Clarke RB.

"A common theme of many investigations into CSCs is that they have inherent resistance to chemo and radiotherapy. This is proposed to be due to mechanisms such as more efficient DNA damage checkpoints and survival pathways compared to more differentiated tumor
cell populations."

"Enhanced interaction between estrogen receptor signalling and growth factor tyrosine kinase pathways such as EGFR, HER2/erbB2 and IGFR mediates resistance to endocrine therapy"

"HDAC inhibitors are being used in a number of on going clinical trials including a phase II trial evaluating vorinostat in ER positive patients with metastatic breast cancer who failed prior aromatase inhibitor therapy and up to three chemotherapy regimes [95]. A report of preliminary findings presented at ASCO 2008 (http://tinyurl.com/ASCO-Vorino-Tam) showed that out of the 17 enrolled patients 21% had a partial response and 29% had stable disease after treatment with vorinostat 400 mg daily for 3 of 4 weeks and tamoxifen 20 mg daily, continuously. These findings suggest that the addition of an HDAC inhibitor to tamoxifen in patients who have failed prior aromatase inhibitors or adjuvant tamoxifen may restore hormone sensitivity."
Final results:
Results: To date, 19 patients [median age 56 years (36-71)] have been enrolled and 17 patients were evaluated for response. Two patients (2/18, 11%) had a pulmonary embolus./DVT. Three patients (3/18, 17%) had Grade 3 fatigue, and one patient each (1/18, 6%) had diarrhea, nausea/vomiting, weight loss, AST/ALT elevations, neutropenia or thrombocytopenia. Predominant Grade 2 toxicities included fatigue, nausea/vomiting, anorexia, bleeding and myelosuppression. Four patients had an objective response (1 complete, 3 partial responses: 4/17, 24%), of those 50% had prior chemotherapy and 75% had failed two aromatase inhibitors for metastatic disease, 50% had failed prior tamoxifen. One additional patient had stable disease for 12 months.
Ongoing phase II (same dosage) at Moffitt:
http://www.clinicaltrial.gov/ct2/show/NCT00365599
"Patients with evidence of visceral crisis are not eligible for this study"


Biological Determinants of Endocrine Resistance in Breast Cancer
(Many sections at link)
http://www.medscape.com/viewarticle/708394
Elizabeth A. Musgrove; Robert L. Sutherland
Published: 09/24/2009

Abstract
Endocrine therapies targeting oestrogen action (anti-oestrogens, such as tamoxifen, and aromatase inhibitors) decrease mortality from breast cancer, but their efficacy is limited by intrinsic and acquired therapeutic resistance. Candidate molecular biomarkers and gene expression signatures of tamoxifen response emphasize the importance of deregulation of proliferation and survival signalling in endocrine resistance.