Distinct Pathways Regulated by RET and Estrogen Receptor in Luminal Breast Cancer Demonstrate the Biological Basis for Combination Therapy.

Department of Surgery, University of Iowa, Iowa City.
Annals of surgery (Impact Factor: 8.33). 09/2013; 259(4). DOI: 10.1097/SLA.0b013e3182a6f552
Source: PubMed


We investigated directed therapy based on TFAP2C-regulated pathways to inform new therapeutic approaches for treatment of luminal breast cancer.
TFAP2C regulates the expression of genes characterizing the luminal phenotype including ESR1 and RET, but pathway cross talk and potential for distinct elements have not been characterized.
Activation of extracellular signal-regulated kinases (ERK) and AKT was assessed using phosphorylation-specific Western blot. Cell proliferation was measured with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] after siRNA (small interfering RNA) gene knockdown or drug treatment. Cell cycle, Ki-67, and cleaved caspase 3 were measured by fluorescence-activated cell sorting. Tumorigenesis was assessed in mice xenografts.
Knockdown of TFAP2C or RET inhibited GDNF (glial cell line-derived neurotrophic factor)-mediated activation of ERK and AKT in MCF-7 cells. Similarly, sunitinib, a small-molecule inhibitor of RET, blocked GDNF-mediated activation of ERK and AKT. Inhibition of RET either by gene knockdown or by treatment with sunitinib or vandetanib reduced RET-dependent growth of luminal breast cancer cells. Interestingly, knockdown of TFAP2C, which controls both ER (estrogen receptor) and RET, demonstrated a greater effect on cell growth than either RET or ER alone. Parallel experiments using treatment with tamoxifen and sunitinib confirmed the increased effectiveness of dual inhibition of the ER and RET pathways in regulating cell growth. Whereas targeting the ER pathway altered cell proliferation, as measured by Ki-67 and S-phase, anti-RET primarily increased apoptosis, as demonstrated by cleaved caspase 3 and increased TUNEL (terminal deoxyneucleotidyl transferase dUTP nick end labeling) expression in xenografts.
ER and RET primarily function through distinct pathways regulating proliferation and cell survival, respectively. The findings inform a therapeutic approach based on combination therapy with antiestrogen and anti-RET in luminal breast cancer.

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    ABSTRACT: Recent findings suggest that combination treatment with anti-estrogen and anti-RET may offer a novel treatment strategy in a subset of breast cancer patients. We investigated the role of RET in potentiating the effects of anti-estrogen response and examined whether RET expression predicted the ability for tyrosine kinase inhibitor (TKI) to effect ERK1/2 activation in primary breast cancer. Growth response, ERK1/2 activation, Ki-67 and TUNEL was assessed in breast cancer cell lines in vitro and in xenografts with vandetanib and/or tamoxifen. Thirty tumors with matched normal breast tissue were evaluated for RET expression and response to TKI treatment. Vandetanib potentiated the inhibitory effect of tamoxifen in hormone responsive (p=0.01) and hormone insensitive (p<0.001) ERα-positive breast cancer cells. Vandetanib significantly repressed tumorigenesis of MCF-7 xenografts (p<0.001), which displayed decreased activation of ERK1/2 and AKT. Vandetanib and tamoxifen reduced the growth of established tumors with a greater effect of dual therapy compared to single agent (p=0.003), with tamoxifen reducing proliferative index and vandetanib inducing apoptosis. In primary breast cancers, RET expression correlated with the ERα-positive subtype. Relative decrease in ERK1/2 phosphorylation with TKI treatment was 42% (p<0.001) in RET-positive tumors vs. 14% (p=ns) in RET-negative tumors. Vandetanib potentiated the anti-growth effects of tamoxifen in breast cancer, which was mediated through RET activation. RET predicted response to TKI therapy with minimal effects on ERK1/2 activation in RET-negative tumors. The preclinical data support evaluation of anti-estrogen in combination with TKI as a potential treatment strategy for RET-positive luminal breast cancer.
    Clinical Cancer Research 02/2014; 20(8). DOI:10.1158/1078-0432.CCR-13-2221 · 8.72 Impact Factor
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    ABSTRACT: De-regulation of RET signalling by oncogenic mutation, gene rearrangement, overexpression or transcriptional up-regulation is implicated in several human cancers of neuroendocrine and epithelial origin (thyroid, breast, lung). Understanding how RET signalling mechanisms associated with these oncogenic events are deregulated, and their impact in the biological processes driving tumor formation and progression, as well as response to treatment, will be crucial to find and develop better targeted therapeutic strategies. In this review we make special emphasis on the distinct mechanisms of RET signaling in cancer and in the current knowledge related to small molecule inhibitors targeting the tyrosine kinase domain of RET as therapeutic drugs in RET-positive cancers.
    Cellular Signalling 04/2014; 26(8). DOI:10.1016/j.cellsig.2014.03.032 · 4.32 Impact Factor

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