p53 status influences response to tamoxifen but not to fulvestrant in breast cancer cell lines.

Mutations in the tumor suppressor gene TP53 are associated with poor prognosis in breast cancer. This prognostic value may rely in part on the fact that p53 plays a role in the antiproliferative and apoptotic activities of chemotherapy regimens used to treat breast tumors. However, some studies suggested that p53 may also influence response to antihormone treatments. Here we investigate how p53 may affect response to antihormonal treatments, using estrogen-dependent breast cancer cell-lines with different p53 status. We show that p53 mutated cells were more resistant to cytotoxic effects of 4-hydroxy-tamoxifen (OHT) compared to p53 wild-type cells. In contrast, p53 status did not significantly impact on response to fulvestrant. p53 mutated cells were also hypersensitive to proliferative effects of estradiol. Interestingly, OHT at doses in the low range had proliferative activities in p53 mutated cells (120-150% proliferation rate under 1 µM OHT treatment in low estrogen conditions). Using gene silencing or specific tyrosine kinase inhibitors, we show that the proliferative effects of OHT were estrogen receptor dependent and could be abrogated by the inhibition of EGFR and/or HER2 kinases. These findings suggest that loss of p53 function may increase cross-talks between estrogen receptor and EGFR/HER2 pathways, contributing to a proliferative effect of OHT. These results bring new insights into the prognostic role of p53 in breast cancer and into possible mechanisms underlying tamoxifen resistance.

Estrogen levels act as a rheostat on p53 levels and modulate p53-dependent responses in breast cancer cell lines.

A large proportion of breast cancers expresses the estrogen receptor alpha (ERα) and are dependent on estrogens for their proliferation and survival. The tumor suppressor TP53 encodes the p53 protein, an important mediator of the anti-proliferative and apoptotic effects of several treatments used for breast cancer. A significant proportions of breast tumors (20-30%) carry mutations in TP53 gene and these mutations are associated with poor survival and poor response to several types of chemotherapeutic treatments. While there is mounting evidence for functional interactions between p53 and ERα pathways in breast and other tissues, the impact of these interactions on response to chemotherapy and anti-hormone treatments remain largely unknown. Here, using estrogen-dependent breast cancer cell lines with different p53 status, we show that estrogens, through ERα, influence p53 protein levels and activities. Estrogen deprivation reduced, while estradiol increased p53 levels, in a time and dose-dependent manner. Both wild-type and endogenously expressed mutant p53 proteins were affected. This reduction in p53 protein levels resulted in reduced p53-dependent responses induced by DNA damage in p53 wild-type cells, lowering the capacity of doxorubicine to induce apoptosis. The p53 response appeared to be quantitatively but not qualitatively affected. These results suggest that ERα activity is required for a strong p53 response in estrogen-dependent breast cancer cells. These results are in line with previous observations that we made in a clinical series, where a larger effect of TP53 mutation status was found for patient survival in cases with progesterone receptor positive status, a marker of a functional ERα pathway. It would thus be important to further characterize the influence of ERα pathway on the predictive value of TP53 mutation status in specifically designed clinical trials, as it may open perspectives for improving breast cancer treatment.

Identification of tyrosine 806 as a molecular determinant of RET kinase sensitivity to ZD6474.

ZD6474 (vandetanib, Zactima, Astra Zeneca) is an anilinoquinazoline used to target the receptor tyrosine kinase RET in familial and sporadic thyroid carcinoma (IC(50): 100 nM). The aim of this study was to identify molecular determinants of RET sensitivity to ZD6474. Here, we show that mutation of RET tyrosine 806 to cysteine (Y806C) induced RET kinase resistance to ZD6474 (IC(50): 933 nM). Y806 maps close to the gate-keeper position at the RET kinase nucleotide-binding pocket. Although tyrosine 806 is a RET auto-phosphorylation site, its substitution to phenylalanine (Y806F) did not markedly affect RET susceptibility to ZD6474 (IC(50): 87 nM), suggesting that phosphorylation of Y806 is not required for compound binding. Accordingly, the introduction of a phosphomimetic residue (Y806E) also caused resistance to ZD6474, albeit of a lesser degree (IC(50): 512 nM) than the cysteine mutation. Y806C/E RET mutants were also resistant to ZD6474 with respect to intracellular signalling and activation of an AP1-responsive promoter. We conclude that Y806 is a molecular determinant of RET sensitivity to ZD6474. Y806C is a natural RET mutation identified in a patient affected by multiple endocrine neoplasia type 2B. Based on its rare occurrence, it is unlikely that Y806C will be a frequent cause of refractoriness to ZD6474; however, it may be envisaged that mutations at this site can mediate secondary resistance formation in patients treated with the compound.

Non-AUG translational initiation of a short CAPC transcript generating protein isoform.

CAPC (also known as LRRC26) is a new gene with restricted expression in normal tissues, and with expression in many cancers and cancer cell lines. We have identified and characterized a short-transcript of CAPC (S-CAPC). The nucleotide sequence analysis of CAPC mRNA showed that the transcription for S-CAPC starts at position +610 on the L-CAPC transcript. Interestingly, no translation initiation codon 'AUG' is present in this transcript. To determine if a non-AUG start site is utilized, the S-CAPC sequence was cloned into an expression vector with C-terminal myc and histidine tags, and transfected into 293T cells. Western blot and MALDI-TOF MS analysis on purified S-CAPC gave two distinct peaks at approximately 7.5 kDa. N-terminal amino acid sequencing of the purified 7.5 kDa protein product indicated that translation starts at the codon for cysteine on the S

Sorafenib inhibits imatinib-resistant KIT and platelet-derived growth factor receptor beta gatekeeper mutants.

PURPOSE: Targeting of KIT and platelet-derived growth factor receptor (PDGFR) tyrosine kinases by imatinib is an effective anticancer strategy. However, mutations of the gatekeeper residue (T670 in KIT and T681 in PDGFRbeta) render the two kinases resistant to imatinib. The aim of this study was to evaluate whether sorafenib (BAY 43-9006), a multitargeted ATP-competitive inhibitor of KIT and PDGFR, was active against imatinib-resistant KIT and PDGFRbeta kinases. EXPERIMENTAL DESIGN: We used in vitro kinase assays and immunoblot with phosphospecific antibodies to determine the activity of sorafenib on KIT and PDGFRbeta kinases. We also exploited reporter luciferase assays to measure the effects of sorafenib on KIT and PDGFRbeta downstream signaling events. The activity of sorafenib on interleukin-3-independent proliferation of Ba/F3 cells expressing oncogenic KIT or its imatinib-resistant T670I mutant was also tested. RESULTS: Sorafenib efficiently inhibited gatekeeper mutants of KIT and PDGFRbeta (IC(50) for KIT T670I, 60 nmol/L; IC(50) for PDGFRbeta T681I, 110 nmol/L). Instead, it was less active against activation loop mutants of the two receptors (IC(50) for KIT D816V, 3.8 micromol/L; IC(50) for PDGFRbeta D850V, 1.17 micromol/L) that are also imatinib-resistant. Sorafenib blocked receptor autophosphorylation and signaling of KIT and PDGFRbeta gatekeeper mutants in intact cells as well as activation of AP1-responsive and cyclin D1 gene promoters, respectively. Finally, the compound inhibited KIT-dependent proliferation of Ba/F3 cells expressing the oncogenic KIT mutant carrying the T670I mutation. CONCLUSIONS: Sorafenib might be a promising anticancer agent for patients carrying KIT and PDGFRbeta gatekeeper mutations.

Clinical case seminar: in vivo and in vitro characterization of a novel germline RET mutation associated with low-penetrant nonaggressive familial medullary thyroid carcinoma.

CONTEXT: RET mutation analysis provides useful information on the clinical outcome of medullary thyroid carcinomas (MTCs) and the risk of disease in the family members. OBJECTIVE: The objective of this study was to document genotype-phenotype relationships in an Italian family with a novel RET mutation. DESIGN/SETTING: RET gene alterations were investigated in a patient with unifocal MTC and her relatives. The identified mutation was subjected to in vitro functional testing. PATIENTS: Patients included a female proband who developed MTC at age 60, her five children, and three grandchildren. MAIN OUTCOME MEASURES: DNA extracted from the blood and the proband's tumor were analyzed for RET alterations. The transforming potential and mitogenic properties of the identified mutation were investigated. RESULTS: A novel heterozygous germline RET mutation at codon 777 (AAC-->AGC, N-->S) (RET/N777S) was identified in the proband and three of her relatives. Two of the latter presented thyroid nodules, but none had MTC or C cell hyperplasia. The proband's MTC was characterized by late onset and limited aggressiveness, with no evidence of regional lymph node or distant metastases 10 yr after total thyroidectomy. This phenotype is consistent with the RET/N777S mutant's low-grade transforming potential and limited activation of RET tyrosine kinase. CONCLUSION: Our findings indicate that the newly identified RET/N777S mutation is a low-penetrant cause of MTC disease. This phenotype might be less aggressive than that associated with MEN2A of familial MTC, although close clinical follow-up of carriers is essential.

Disease associated mutations at valine 804 in the RET receptor tyrosine kinase confer resistance to selective kinase inhibitors.

We have recently demonstrated that the pyrazolopyrimidines PP1 and PP2 and the 4-anilinoquinazoline ZD6474 display a strong inhibitory activity (IC(50)< or =100 nM) towards constitutively active oncogenic RET kinases. Here, we show that most oncogenic MEN2-associated RET kinase mutants are highly susceptible to PP1, PP2 and ZD6474 inhibition. In contrast, MEN2-associated swap of bulky hydrophobic leucine or methionine residues for valine 804 in the RET kinase domain causes resistance to the three compounds. Substitution of valine 804 with the small amino- acid glycine renders the RET kinase even more susceptible to inhibition (ZD6474 IC(50): 20 nM) than the wild-type kinase. Our data identify valine 804 of RET as a structural determinant mediating resistance to pyrazolopyrimidines and 4-anilinoquinazolines.

Functional characterization of a novel FGFR1OP-RET rearrangement in hematopoietic malignancies.

The RET (REarranged during Transfection) receptor tyrosine kinase is targeted by oncogenic rearrangements in thyroid and lung adenocarcinoma. Recently, a RET (exon 12) rearrangement with FGFR1OP [fibroblast growth factor receptor 1 (FGFR1) oncogene partner] (exon 12) was identified in one chronic myelomonocytic leukemia (CMML) patient. We report the molecular cloning and functional characterization of a novel FGFR1OP (exon 11)-RET (exon 11) gene fusion event (named FGFR1OP-RET), mediated by a reciprocal translocation t(6; 10)(q27; q11), in a patient affected by primary myelofibrosis (PMF) with secondary acute myeloid leukemia (AML). The FGFR1OP-RET fusion protein displayed constitutive tyrosine kinase and transforming activity in NIH3T3 fibroblasts, and induced IL3-independent growth and activation of PI3K/STAT signaling in hematopoietic Ba/F3 cells. FGFR1OP-RET supported cytokine-independent growth, protection from stress and enhanced self-renewal of primary murine hematopoietic progenitor and stem cells in vitro. In vivo, FGFR1OP-RET caused a spectrum of disease phenotypes, with >50% of mice showing a fatal myeloproliferative disorder (MPD). Other phenotypes were leukemia transplantable in secondary recipients, dramatic expansion of the mast cell lineage, and reduction of repopulating activity upon lethal irradiation. In conclusion, FGFR1OP-RET chimeric oncogenes are endowed with leukemogenic potential and associated to myeloid neoplasms (CMML and PMF/AML).

p53-Dependent repression of focal adhesion kinase in response to estradiol in breast cancer cell-lines.

Mutations in the TP53 suppressor gene are frequent in breast cancers. These mutations are associated with poor prognosis, thought to be due to proliferative advantage and poor response to chemotherapy associated with loss of p53 function. The focal adhesion kinase (FAK/PTK2), a tyrosine kinase, is over-expressed in a variety of human tumors including breast cancers. FAK is a critical regulator of adhesion and motility and its over-expression is associated with increased metastatic potential. Recently, FAK promoter has been shown to contain p53 responsive elements and to be down-regulated by DNA-damage in a p53-dependent manner. Here, we have used five estrogen-dependent breast cancer cells lines with different p53 status, including an isogenic model, to show that FAK expression was regulated in a p53-dependent manner in response to estradiol. FAK protein and mRNA expression were down-regulated by estradiol in wild-type but not mutant p53 cells. Moreover, silencing wild-type p53 increased FAK expression, while over expressing p53 repressed FAK expression. ChIP experiment showed that p53 bound to FAK promoter in the presence of estradiol in p53 wild-type but not in mutant p53 cells, suggesting a direct role of p53 in down regulating FAK mRNA expression. FAK mRNA expression was also found to correlate with TP53 mutation status in a series of breast tumors. Finally, loss of FAK down-regulation in p53 mutant cells was correlated with increased proliferation and invasion upon estradiol stimulation, while FAK silencing reduced invasion. These results suggest that p53 is an important down regulator of FAK and that loss of p53 function in breast cancer may contribute to the metastatic potential of estrogen-responsive tumors through uncontrolled FAK expression upon estrogens stimulation.

BAY 43-9006 inhibition of oncogenic RET mutants.

BACKGROUND: Medullary and papillary thyroid carcinomas are often associated with oncogenic activation of the RET tyrosine kinase. We evaluated whether the biaryl urea BAY 43-9006, which is known to inhibit several other tyrosine kinases, blocks RET kinase function and oncogenic activity. METHODS: We examined BAY 43-9006 activity against oncogenic RET in vitro and in cellular RET signaling in oncogenic RET-transfected NIH3T3 fibroblasts by using immunocomplex kinase assays and immunoblotting with phospho-specific antibodies. The effects of BAY 43-9006 on proliferation of human TPC1 and TT thyroid carcinoma cells, which harbor spontaneous oncogenic RET alleles, and on RAT1 fibroblasts transformed with oncogenic RET mutants, including mutants that are resistant to other chemotherapeutic agents, were determined using growth curves and flow cytometry. Growth of TT cell-derived xenograft tumors in athymic mice treated orally with BAY 43-9006 or with vehicle was measured. All statistical tests were two-sided. RESULTS: BAY 43-9006 inhibited oncogenic RET kinase activity at half-maximal inhibitory concentrations (IC50s) of 50 nM or less in NIH3T3 cells. It also arrested the growth of NIH3T3 and RAT1 fibroblasts transformed by oncogenic RET and of thyroid carcinoma cells that harbor spontaneous oncogenic RET alleles. Moreover, BAY 43-9006 inhibited the growth of cells carrying RET V804L (IC50 = 110 nM, 95% confidence interval [CI] = 88 to 133 nM) or RET V804M (IC50 = 147 nM, 95% CI = 123 nM to 170 nM), both mutants that are resistant to anilinoquinazolines and pyrazolopyrimidines. After 3 weeks of oral treatment with BAY 43-9006 (60 mg/kg/day), the volume of TT cell xenografts (n = 7) was reduced from 72.5 to 44 mm3 (difference = 28.5 mm3, 95% CI = 7 mm3 to 50 mm3), whereas in vehicle-treated mice (n = 7), mean tumor volume increased to 408 mm3 (difference = 320 mm3, 95% CI = 180 mm3 to 460 mm3; untreated versus treated, P =.02). This inhibition paralleled a decrease in RET phosphorylation. CONCLUSIONS: BAY 43-9006 is a powerful inhibitor of the RET kinase. Its potential as a therapeutic tool for RET-positive thyroid tumors, including those expressing V804 mutations merits study.