Maria Graciela Olivares

Acquired resistance to EGFR tyrosine kinase inhibitors in cancer cells is mediated by loss of IGF-binding proteins

Although some cancers are initially sensitive to EGFR tyrosine kinase inhibitors (TKIs), resistance invariably develops. We investigated mechanisms of acquired resistance to the EGFR TKI gefitinib by generating gefitinib-resistant (GR) A431 squamous cancer cells. In GR cells, gefitinib reduced phosphorylation of EGFR, ErbB-3, and Erk but not Akt. These cells also showed hyperphosphorylation of the IGFI receptor (IGFIR) and constitutive association of IRS-1 with PI3K. Inhibition of IGFIR signaling disrupted the association of IRS-1 with PI3K and restored the ability of gefitinib to downregulate PI3K/Akt signaling and to inhibit GR cell growth. Gene expression analyses revealed that GR cells exhibited markedly reduced IGF-binding protein 3 (IGFBP-3) and IGFBP-4 RNA. Addition of recombinant IGFBP-3 restored the ability of gefitinib to downregulate PI3K/Akt signaling and to inhibit cell growth. Finally, gefitinib treatment of mice with A431 xenografts in combination with an IGFIR-specific monoclonal antibody prevented tumor recurrence, whereas each drug given alone was unable to do so. These data suggest that loss of expression of IGFBPs in tumor cells treated with EGFR TKIs derepresses IGFIR signaling, which in turn mediates resistance to EGFR antagonists. Moreover, combined therapeutic inhibition of EGFR and IGFIR may abrogate this acquired mechanism of drug resistance and is thus worthy of prospective clinical investigation.

Transcriptional and posttranslational up-regulation of HER3 (ErbB3) compensates for inhibition of the HER2 tyrosine kinase

Sustained and complete inhibition of HER3 and its output to PI3K/Akt are required for the optimal antitumor effect of therapeutic inhibitors of the HER2 oncogene. Here, we show that, after inhibition of the HER2 tyrosine kinase with lapatinib, there is PI3K/Akt and FoxO3a-dependent up-regulation of HER3 mRNA and protein. Up-regulated HER3 was then phosphorylated by residual HER2 activity, thus partially maintaining P-Akt and limiting the antitumor action of lapatinib. Inhibition of HER3 with siRNA or a neutralizing HER3 antibody sensitized HER2+ breast cancer cells and xenografts to lapatinib both in vitro and in vivo. Combined blockade of HER2 and HER3 inhibited pharmacodynamic biomarkers of PI3K/Akt activity more effectively than each inhibitor alone. These results suggest that because of HER3-mediated compensation, current clinical inhibitors of HER2 and PI3K/Akt will not block the PI3K pathway completely. They also suggest that therapeutic inhibitors of HER3 should be used in combination with HER2 inhibitors and PI3K pathway inhibitors in patients with HER2- and PI3K-dependent cancers.

Transforming Growth Factor β Engages TACE and ErbB3 To Activate Phosphatidylinositol-3 Kinase/Akt in ErbB2-Overexpressing Breast Cancer and Desensitizes Cells to Trastuzumab

ABSTRACT In HER2-overexpressing mammary epithelial cells, transforming growth factor β (TGF-β) activated phosphatidylinositol-3 kinase (PI3K)/Akt and enhanced survival and migration. Treatment with TGF-β or expression of an activated TGF-β type I receptor (Alk5 with the mutation T204D [Alk5T204D]) induced phosphorylation of TACE/ADAM17 and its translocation to the cell surface, resulting in increased secretion of TGF-α, amphiregulin, and heregulin. In turn, these ligands enhanced the association of p85 with ErbB3 and activated PI3K/Akt. RNA interference of TACE or ErbB3 prevented TGF-β-induced activation of Akt and cell invasiveness. Treatment with TGF-β or expression of Alk5T204D in HER2-overexpressing cells reduced their sensitivity to the HER2 antibody trastuzumab. Inhibition of Alk5, PI3K, TACE, or ErbB3 restored sensitivity to trastuzumab. A gene signature induced by Alk5T204D expression correlated with poor clinical outcomes in patients with invasive breast cancer. These results suggest that by acting on ErbB ligand shedding, an excess of TGF-β may result in (i) conditioning of the tumor microenvironment with growth factors that can engage adjacent stromal and endothelial cells; (ii) potentiation of signaling downstream ErbB receptors, thus contributing to tumor progression and resistance to anti-HER2 therapies; and (iii) poor clinical outcomes in women with breast cancer.

Identification of markers of taxane sensitivity using proteomic and genomic analyses of breast tumors from patients receiving neoadjuvant paclitaxel and radiation.

Purpose: To identify molecular markers of pathologic response to neoadjuvant paclitaxel/radiation treatment, protein and gene expression profiling were done on pretreatment biopsies. Experimental Design: Patients with high-risk, operable breast cancer were treated with three cycles of paclitaxel followed by concurrent paclitaxel/radiation. Tumor tissue from pretreatment biopsies was obtained from 19 of the 38 patients enrolled in the study. Protein and gene expression profiling were done on serial sections of the biopsies from patients that achieved a pathologic complete response (pCR) and compared to those with residual disease, non-pCR (NR). Results: Proteomic and validation immunohistochemical analyses revealed that α-defensins (DEFA) were overexpressed in tumors from patients with a pCR. Gene expression analysis revealed that MAP2, a microtubule-associated protein, had significantly higher levels of expression in patients achieving a pCR. Elevation of MAP2 in breast cancer cell lines led to increased paclitaxel sensitivity. Furthermore, expression of genes that are associated with the basal-like, triple-negative phenotype were enriched in tumors from patients with a pCR. Analysis of a larger panel of tumors from patients receiving presurgical taxane-based treatment showed that DEFA and MAP2 expression as well as histologic features of inflammation were all statistically associated with response to therapy at the time of surgery. Conclusion: We show the utility of molecular profiling of pretreatment biopsies to discover markers of response. Our results suggest the potential use of immune signaling molecules such as DEFA as well as MAP2, a microtubule-associated protein, as tumor markers that associate with response to neoadjuvant taxane–based therapy. Clin Cancer Res; 16(2); 681–90

Inhibition of Mammalian Target of Rapamycin Is Required for Optimal Antitumor Effect of HER2 Inhibitors against HER2-Overexpressing Cancer Cells

Purpose: A significant fraction of HER2-overexpressing breast cancers exhibit resistance to the HER2 antibody trastuzumab. Hyperactivity of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway confers trastuzumab resistance, and mammalian target of rapamycin (mTOR) is a major downstream effector of PI3K/AKT. Therefore, we examined whether mTOR inhibitors synergize with trastuzumab. Experimental Design: Immunocompetent mice bearing HER2+ mammary tumors were treated with trastuzumab, the mTOR inhibitor rapamycin, or the combination. Mice were imaged for tumor cell death using an optical Annexin-V probe and with [18F]FDG positron emission tomography. The signaling and growth effects of the mTOR inhibitor RAD001 on HER2+ cells treated with trastuzumab or lapatinib were evaluated. Results: Treatment of mice with trastuzumab plus rapamycin was more effective than single-agent treatments, inducing complete regression of 26 of 26 tumors. The combination induced tumor cell death (Annexin-V binding) and inhibited FDG uptake. Rapamycin inhibited mTOR and tumor cell proliferation as determined by phosphorylated S6 and Ki-67 immunohistochemistry, respectively. In culture, the combination of RAD001 plus trastuzumab inhibited cell growth more effectively than either drug alone. Trastuzumab partially decreased PI3K but not mTOR activity. Knockdown of TSC2 resulted in HER2-independent activation of mTOR and dampened the response to trastuzumab and lapatinib. Treatment with the HER2 inhibitor lapatinib decreased phosphorylated S6 and growth in TSC2-expressing cells but not in TSC2-knockdown cells. Conclusions: Inhibition of PI3K and mTOR are required for the growth-inhibitory effect of HER2 antagonists. These findings collectively support the combined use of trastuzumab and mTOR inhibitors for the treatment of HER2+ breast cancer. (Clin Cancer Res 2009;15(23):7266–76)

Imaging Biomarkers Predict Response to Anti-HER2 (ErbB2) Therapy in Preclinical Models of Breast Cancer

Purpose: To evaluate noninvasive imaging methods as predictive biomarkers of response to trastuzumab in mouse models of HER2-overexpressing breast cancer. The correlation between tumor regression and molecular imaging of apoptosis, glucose metabolism, and cellular proliferation was evaluated longitudinally in responding and nonresponding tumor-bearing cohorts. Experimental Design: Mammary tumors from MMTV/HER2 transgenic female mice were transplanted into syngeneic female mice. BT474 human breast carcinoma cell line xenografts were grown in athymic nude mice. Tumor cell apoptosis (NIR700-Annexin V accumulation), glucose metabolism [2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography ([18F]FDG-PET)], and proliferation [3′-[18F]fluoro-3′-deoxythymidine-PET ([18F]FLT-PET)] were evaluated throughout a biweekly trastuzumab regimen. Imaging metrics were validated by direct measurement of tumor size and immunohistochemical analysis of cleaved caspase-3, phosphorylated AKT, and Ki67. Results: NIR700-Annexin V accumulated significantly in trastuzumab-treated MMTV/HER2 and BT474 tumors that ultimately regressed but not in nonresponding or vehicle-treated tumors. Uptake of [18F]FDG was not affected by trastuzumab treatment in MMTV/HER2 or BT474 tumors. [18F]FLT-PET imaging predicted trastuzumab response in BT474 tumors but not in MMTV/HER2 tumors, which exhibited modest uptake of [18F]FLT. Close agreement was observed between imaging metrics and immunohistochemical analysis. Conclusions: Molecular imaging of apoptosis accurately predicts trastuzumab-induced regression of HER2+ tumors and may warrant clinical exploration to predict early response to neoadjuvant trastuzumab. Trastuzumab does not seem to alter glucose metabolism substantially enough to afford [18F]FDG-PET significant predictive value in this setting. Although promising in one preclinical model, further studies are required to determine the overall value of [18F]FLT-PET as a biomarker of response to trastuzumab in HER2+ breast cancer.

Heritable variation of ERBB2 and breast cancer risk.

Amplification of the epithelial growth factor receptor gene ERBB2 (HER2, NEU) in breast cancer is associated with a poor clinical prognosis. In mammary gland development, this receptor plays a role in ductal and lobuloalveolar differentiation. We conducted a systematic investigation of the role of genetic variation of the ERBB2 gene in breast cancer risk in a study of 842 histologically confirmed invasive breast cancer cases and 1,108 controls from the Shanghai Breast Cancer Study. We observed that the ERBB2 gene resides within a locus of high linkage disequilibrium, composed of three major ancestral haplotypes in the study population. These haplotypes are marked by simple tandem repeat and single nucleotide polymorphisms, including the missense variants I655V and P1170A. We observed a risk-modifying effect of a highly polymorphic simple tandem repeat within an evolutionarily conserved region, 4.4 kb upstream from the ERBB2 transcription start site. Under a dominant genetic model, the age-adjusted odds ratio was 1.74 (95% confidence interval, 1.27-2.37). Its association with breast cancer, and with breast cancer stratified by histology, by histologic grade, and by stage, remained significant after correction for multiple comparisons. In contrast, we observed no association of ERBB2 single nucleotide polymorphism haplotypes with breast cancer predisposition. (Cancer Epidemiol Biomarkers Prev 2009;18(4):1252–8)

Transforming growth factor beta engages TACE/ADAM17 and ErbB3 to activate PI3K/Akt in HER2-overexpressing breast cancer and desensitizes cells to trastuzumab.

Abstract #35 TGFb signaling synergizes with HER2 in breast cancer progression. Exogenous TGFb or expression of an activated TGFb type I receptor (Alk5 T204D ) in MCF10A/HER2 and BT474 cells activated PI3K/Akt and enhanced survival and migration. In both cell types, TGFb stimulated P-EGFR and P-HER2 as well as the association of p85, the regulatory subunit of PI3K, with Y1289 P-ErbB3 which, in turn, activated PI3K/Akt. RNA interference of ErbB3 or treatment with pertuzumab, an antibody that blocks HER2-mediated activation of ErbB3, blocked TGFb-induced P-ErbB3, P-Akt, and cell motility. Treatment with TGFb or expression of Alk5 TD increased protein levels of secreted TGFalpha, amphiregulin, and heregulin without a change in mRNA levels. The increase on secreted ErbB ligands without a change in mRNA levels suggested increased shedding of ErbB pro-ligands by tumor necrosis factor a-converting enzyme (TACE). Transfection of siRNA against human TACE but not control siRNA abrogated TGFb-stimulated ErbB receptor phosphorylation, P-Akt, and cell invasiveness. Transfection of full-length mouse TACE but not truncated TACE lacking the cytoplasmic domain reconstituted TGFb-induced ErbB phosphorylation in cells transfected with human TACE siRNA. TGF-b increased TACE phosphorylation in both serine and threonine as measured by P-Ser and P-Thr immunoblots of TACE pull downs. Incubation of TACE with Alk5 TD in the presence of ATP in vitro resulted in both Ser and Thr phosphorylation in full-length but not truncated TACE. Further, addition of TGFb or expression of Alk5 TD desensitized SKBR3 and BT474 cells to trastuzumab. Treatment with the type I TGFb receptor (Alk5) small molecule inhibitor LY2109761, the PI3K inhibitor LY294002, or siRNA against ErbB3 or TACE restored the inhibitory effect of trastuzumab. Finally, we generated an active Alk5 expression signature by selecting genes that were differentially expressed between BT474/Alk5 TD and BT474/vector cells. Mapping of this signature to a previously published 295-breast tumor array (van de Vijver et al. and Chang et al. ) revealed 90 of 271 genes in the Alk5 TD signature with a >1.5-fold change from the median expression which correlated with HER2-positive and basal-like tumors and conferred a worse recurrence-free (RFS) and overall survival (OS) (RFS p =0.05, OS p =0.01). Hierarchical clustering analysis to an array data set reported by Harris et al. in 22 patients with HER2+ breast cancer treated with neoadjuvant trastuzumab and vinorelbine ( Clin. Cancer Res. 2007) showed that the Alk5 TD signature correlated with tumors that did not respond clinically. These data suggest that TGFb potentiates signaling downstream ErbB receptors via TACE, ErbB3, and PI3K, thus contributing to tumor progression and resistance to anti-HER2 therapies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 35.

Transcriptional and Post-Translational Upregulation of HER3 (ErbB3) Counteracts Antitumor Effect of HER2 Tyrosine Kinase Inhibitors.

We hypothesized that sustained inhibition of HER3 and its output to PI3K/Akt is required for the optimal antitumor effect of HER2 inhibitors. Therefore, we examined the temporal effect of the HER2 tyrosine kinase inhibitor (TKI) lapatinib (lap) on feedback upregulation of active HER3 in HER2-overexpressing breast cancer cells. A time course with lap-treated cells showed 3 to 5-fold upregulation of HER3 RNA and protein, beginning at 4 h and increasing through 48 h. P-Tyr immunoblot of HER3 immunoprecipitates revealed recovery of HER3 phosphorylation at and beyond 13 h of treatment. Site-specific antibodies revealed HER3 phosphorylation at Y1197 and Y1289, two of the six p85 binding sites in HER3. Recovery of P-HER3 correlated temporally with recovery of T308 P-Akt. The upregulation of HER3 RNA upon treatment with lap suggested that inhibition of active HER2 and PI3K/Akt derepresses the transcription factor FoxO3a. Putative FoxO3a binding sites were identified within the 59 flanking region upstream of the HER3 transcription start site. Transfection with FoxO3a siRNA reduced basal and lap-induced HER3 RNA levels 2 to 5-fold compared to control cells. Conversely, overexpression of FoxO3a increased HER3 RNA 2.5-fold, which could be further enhanced by lap treatment. In addition to these transcriptional mechanisms, the recovery of P-HER3 upon lap-induced inhibition of HER2 suggested engagement of another tyrosine kinase transactivating HER3 and/or that HER2 had been incompletely inhibited by the TKI. However, IGF-IR, Src, and MET TKIs did not inhibit the recovery of P-HER3. On the other hand, the addition of trastuzumab (tz) to lap-treated cells prevented recovery of P-HER3, suggesting that disruption of a ligand-independent HER2-HER3 interaction was involved in partial maintenance of HER3 phosphorylation.The upregulation of HER3 RNA and partial maintenance of P-HER3 and P-Akt suggested that combined inhibition of HER2 and HER3 will synergistically inhibit tumor cell viability. Transfection with HER3 siRNA sensitized HER2+ breast cancer cells to each lap and tz as assessed by Apo-BrdU (apoptosis) and 3D-Matrigel growth assays. Further, treatment with AMG-888, a HER3 monoclonal antibody (AMGEN-U3), sensitized cells to each lap and tz. Ongoing studies include the treatment of BT474 xenografts in athymic mice with lap ± AMG-888 using [ 18 F]-FDG-PET as a non-invasive imaging biomarker to predict treatment outcome. Finally, we examined HER3 levels by immunohistochemistry in sections from tumor blocks of patients enrolled in a neoadjuvant trial where lap was given alone during the first 6 weeks of therapy. The percent and intensity of tumor cell staining was calculated as a histoscore (Human Pathol. 26:291, 1995). On week 2 of therapy, HER3 levels increased 135% above pre-therapy levels (n=8; p=0.03, Mann-Whitney). These data suggest that upon inhibition of the HER2 tyrosine kinase, HER2+ breast cancers 1) upregulate HER3 by transcriptional mechanisms and partially maintain HER3 function by post-translational mechanisms; 2) this compensatory phosphorylation of HER3 partially maintains PI3K/Akt; and 3) inhibition of HER3 sensitizes HER2-dependent breast cancer cells to HER2 inhibitors. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 63.

Abstract PR-6: Src family kinases mediate PI3K‐Akt pathway activation and resistance to lapatinib in HER2‐overexpressing human breast cancer cells

We investigated mechanisms of resistance to the HER2 tyrosine kinase inhibitor (TKI) lapatinib (Lap) in breast cancer cells that overexpress HER2. Inhibition of phosphoinositide‐3 kinase (PI3K)‐Akt signaling downstream of HER2 has been proposed as required for the antitumor activity of HER2 inhibitors. Resistance to HER2 inhibitors has been associated with aberrant activation of the PI3K pathway. HER2‐overexpressing breast cancer cells (BT‐474, SKBR‐3, HCC1954, MDA‐MB‐361, SUM190, and UACC893) were made resistant to Lap by exposure to increasing concentrations of the drug. Lapatinib‐resistant (LR) cells exhibited the same level of HER2 gene amplification as parental, sensitive cells as measured by fluorescent in situ hybridization. HER2 activity measured by Y1248 P‐HER2 immunoblots remained inhibited in LR cells. However, they consistently showed recovery of PI3K‐Akt activity as demonstrated by phosphorylation of Akt at Ser473. We hypothesized that resistance to HER2 inhibitors results from reactivation of the PI3K‐Akt axis through upregulation of alternate compensatory signaling pathways. To identify these putative escape pathways, we profiled the tyrosine (tyr) phosphoproteome using an immunoaffinity method of purification of tyr‐phosphorylated peptides and identification by tandem liquid chromatography‐mass spectrometry (LC‐MS). Cell lysates from LR and sensitive cells were digested with trypsin and precipitated with a P‐tyr antibody followed by tandem LC‐MS. Peptide sequences were assigned from mass spectra using Myrimatch software and protein identities were determined by analysis with IDPicker. In this analysis, 820 spectra corresponding to 173 tyr‐phosphorylated peptides were identified. Of these, 29 peptides were isolated more frequently or uniquely in LR cells based on spectral counts. To identify kinases that might be responsible for the unique phosphopeptides in the resistant cells, we used Kinase Enrichment Analysis with the 21 protein substrates from these 29 peptides. This analysis suggested that Src or a Src family kinase (SFK) activity was enriched in LR cells. By western blot, we observed increased Y416 P‐SFK in four of the LR lines: BT‐474, MDA‐MB‐361, HCC1954, and UACC893. The MS phosphotyrosine profiling identified Yes in BT‐474 LR cells. This was confirmed by western analysis with Yes specific antibodies and qRT‐PCR with SFK‐specific primers. In HCC1954 cells, western analysis with Src and Lyn antibodies and qRT‐PCR suggested that Lyn and/or Src are the active kinases, whereas Yes was the predominant SFK in MDA‐MB‐361 and UACC893 cells. Treatment of LR cells with the small molecule Src inhibitors AZD0530 or dasatinib in combination with Lap resulted in inhibition of Y416 P‐Src, partial inhibition of S473 P‐Akt, and inhibition of cell growth. Treatment of HCC1954 or MDA‐MB‐361 sensitive cells with Src inhibitors and Lap for 21 days partially abrogated the emergence of lapatinib‐resistant colonies. These data suggest that combination of Src inhibitors with Lap will prevent or overcome therapeutic resistance to the HER2 TKI. Studies to determine 1) the mechanism whereby PI3K‐Akt is activated by SFKs in the resistant cells, and 2) the synergistic effect of combination treatment with Src inhibitors and lapatinib against HER2‐overexpressing xenografts are ongoing at this time. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):PR-6.