Anindita Chakrabarty

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.

Feedback upregulation of HER3 (ErbB3) expression and activity attenuates antitumor effect of PI3K inhibitors

We examined the effects of an inhibitor of PI3K, XL147, against human breast cancer cell lines with constitutive PI3K activation. Treatment with XL147 resulted in dose-dependent inhibition of cell growth and levels of pAKT and pS6, signal transducers in the PI3K/AKT/TOR pathway. In HER2-overexpressing cells, inhibition of PI3K was followed by up-regulation of expression and phosphorylation of multiple receptor tyrosine kinases, including HER3. Knockdown of FoxO1 and FoxO3a transcription factors suppressed the induction of HER3, InsR, IGF1R, and FGFR2 mRNAs upon inhibition of PI3K. In HER2+ cells, knockdown of HER3 with siRNA or cotreatment with the HER2 inhibitors trastuzumab or lapatinib enhanced XL147-induced cell death and inhibition of pAKT and pS6. Trastuzumab and lapatinib each synergized with XL147 for inhibition of pAKT and growth of established BT474 xenografts. These data suggest that PI3K antagonists will inhibit AKT and relieve suppression of receptor tyrosine kinase expression and their activity. Relief of this feedback limits the sustained inhibition of the PI3K/AKT pathway and attenuates the response to these agents. As a result, PI3K pathway inhibitors may have limited clinical activity overall if used as single agents. In patients with HER2-overexpressing breast cancer, PI3K inhibitors should be used in combination with HER2/HER3 antagonists.

MicroRNA regulation of cyclooxygenase-2 during embryo implantation

The implantation process is complex, requiring reciprocal interactions between implantation-competent blastocysts and the receptive uterus. Because microRNAs (miRNAs) have major roles in regulating gene expression, we speculated that they participate in directing the highly regulated spatiotemporally expressed genetic network during implantation. Here, we show that two miRNAs, mmu-miR-101a and mmu-miR-199a*, are spatiotemporally expressed in the mouse uterus during implantation coincident with expression of cyclooxygenase-2, a gene critical for implantation. More interestingly, our in vitro gain- and loss-of-function experiments show that cyclooxygenase-2 expression is posttranscriptionally regulated by these two miRNAs. We report on miRNA-mediated regulation of uterine gene expression in the context of implantation. We believe that many other critical genes related to this process are also regulated by miRNAs. Thus, elucidating the physiological roles of uterine miRNAs will help us better understand the genetic control of implantation, the gateway to a successful pregnancy.

Loss of Phosphatase and Tensin Homolog or Phosphoinositol-3 Kinase Activation and Response to Trastuzumab or Lapatinib in Human Epidermal Growth Factor Receptor 2–Overexpressing Locally Advanced Breast Cancers

PURPOSE Phosphatase and tensin homolog (PTEN) loss or activating mutations of phosphoinositol-3 (PI3) kinase (PIK3CA) may be associated with trastuzumab resistance. Trastuzumab, the humanized human epidermal growth factor receptor 2 (HER2) monoclonal antibody, and lapatinib, an epidermal growth factor receptor/HER2 tyrosine kinase inhibitor, are both established treatments for HER2-overexpressing breast cancers. Understanding of the cellular response to HER2-targeted therapies is needed to tailor treatments and to identify patients less likely to benefit. METHODS We evaluated the effect of trastuzumab or lapatinib in three HER2-overexpressing cell lines. We confirmed the in vitro observations in two neoadjuvant clinical trials in patients with HER2 overexpression; 35 patients received trastuzumab as a single agent for the first 3 weeks, then docetaxel every 3 weeks for 12 weeks (trastuzumab regimen), whereas 49 patients received lapatinib as a single agent for 6 weeks, followed by trastuzumab/docetaxel for 12 weeks before primary surgery (lapatinib regimen). Apoptosis, Ki67, p-MAPK, p-AKT, and PTEN were assessed by immunohistochemistry. Genomic DNA was sequenced for PIK3CA mutations. RESULTS Under low PTEN conditions, in vitro data indicate that lapatinib alone and in combination with trastuzumab was effective in decreasing p-MAPK and p-AKT levels, whereas trastuzumab was ineffective. In the clinical trials, we confirmed that low PTEN or activating mutation in PIK3CA conferred resistance to the trastuzumab regimen (P = .015), whereas low PTEN tumors were associated with a high pathologic complete response rate (P = .007). CONCLUSION Activation of PI3 kinase pathway is associated with trastuzumab resistance, whereas low PTEN predicted for response to lapatinib. These observations support clinical trials with the combination of both agents.

Trastuzumab has preferential activity against breast cancers driven by HER2 homodimers

In breast cancer cells with HER2 gene amplification, HER2 receptors exist on the cell surface as monomers, homodimers, and heterodimers with EGFR/HER3. The therapeutic antibody trastuzumab, an approved therapy for HER2(+) breast cancer, cannot block ligand-induced HER2 heterodimers, suggesting it cannot effectively inhibit HER2 signaling. Hence, HER2 oligomeric states may predict the odds of a clinical response to trastuzumab in HER2-driven tumors. To test this hypothesis, we generated nontransformed human MCF10A mammary epithelial cells stably expressing a chimeric HER2-FKBP molecule that could be conditionally induced to homodimerize by adding the FKBP ligand AP1510, or instead induced to heterodimerize with EGFR or HER3 by adding the heterodimer ligands EGF/TGFα or heregulin. AP1510, EGF, and heregulin each induced growth of MCF10A cells expressing HER2-FKBP. Trastuzumab inhibited homodimer-mediated but not heterodimer-mediated cell growth. In contrast, the HER2 antibody pertuzumab, which blocks HER2 heterodimerization, inhibited growth induced by heregulin but not AP1510. Lastly, the HER2/EGFR tyrosine kinase inhibitor lapatinib blocked both homodimer- and heterodimer-induced growth. AP1510 triggered phosphorylation of Erk1/2 but not AKT, whereas trastuzumab inhibited AP1510-induced Erk1/2 phosphorylation and Shc-HER2 homodimer binding, but not TGFα-induced AKT phosphorylation. Consistent with these observations, high levels of HER2 homodimers correlated with longer time to progression following trastuzumab therapy in a cohort of patients with HER2-overexpressing breast cancer. Together, our findings confirm the notion that HER2 oligomeric states regulate HER2 signaling, also arguing that trastuzumab sensitivity of homodimers may reflect their inability to activate the PI3K (phosphoinositide 3-kinase)/AKT pathway. A clinical implication of our results is that high levels of HER2 homodimers may predict a positive response to trastuzumab.

Trastuzumab-Resistant Cells Rely on a HER2-PI3K-FoxO-Survivin Axis and Are Sensitive to PI3K Inhibitors

The antibody trastuzumab is approved for treatment of patients with HER2 (ERBB2)-overexpressing breast cancer. A significant fraction of these tumors are either intrinsically resistant or acquire resistance rendering the drug ineffective. The development of resistance has been attributed to failure of the antibody to inhibit phosphoinositide 3-kinase (PI3K), which is activated by the HER2 network. Herein, we examined the effects of PI3K blockade in trastuzumab-resistant breast cancer cell lines. Treatment with the pan-PI3K inhibitor XL147 and trastuzumab reduced proliferation and pAKT levels, triggering apoptosis of trastuzumab-resistant cells. Compared with XL147 alone, the combination exhibited a superior antitumor effect against trastuzumab-resistant tumor xenografts. Furthermore, treatment with XL147 and trastuzumab reduced the cancer stem-cell (CSC) fraction within trastuzumab-resistant cells both in vitro and in vivo. These effects were associated with FoxO-mediated inhibition of transcription of the antiapoptosis gene survivin (BIRC5) and the CSC-associated cytokine interleukin-8. RNA interference-mediated or pharmacologic inhibition of survivin restored sensitivity to trastuzumab in resistant cells. In a cohort of patients with HER2-overexpressing breast cancer treated with trastuzumab, higher pretreatment tumor levels of survivin RNA correlated with poor response to therapy. Together, our results suggest that survivin blockade is required for therapeutic responses to trastuzumab and that by combining trastuzumab and PI3K inhibitors, CSCs can be reduced within HER2(+) tumors, potentially preventing acquired resistance to anti-HER2 therapy.

H1047R phosphatidylinositol 3-kinase mutant enhances HER2-mediated transformation by heregulin production and activation of HER3.

Hyperactivation of phosphatidylinositol-3 kinase (PI3K) can occur as a result of somatic mutations in PIK3CA, the gene encoding the p110α subunit of PI3K. The HER2 oncogene is amplified in 25% of all breast cancers and some of these tumors also harbor PIK3CA mutations. We examined mechanisms by which mutant PI3K can enhance transformation and confer resistance to HER2-directed therapies. We introduced the PI3K mutations E545K and H1047R in MCF10A human mammary epithelial cells that also overexpress HER2. Both mutants conferred a gain of function to MCF10A/HER2 cells. Expression of H1047R PI3K, but not E545K PI3K, markedly upregulated the HER3/HER4 ligand heregulin (HRG). HRG siRNA inhibited growth of H1047R but not E545K-expressing cells and synergized with the HER2 inhibitors trastuzumab and lapatinib. The PI3K inhibitor BEZ235 markedly inhibited HRG and pAKT levels and, in combination with lapatinib, completely inhibited growth of cells expressing H1047R PI3K. These observations suggest that PI3K mutants enhance HER2-mediated transformation by amplifying the ligand-induced signaling output of the ErbB network. This also counteracts the full effect of therapeutic inhibitors of HER2. These data also suggest that mammary tumors that contain both HER2 gene amplification and PIK3CA mutations should be treated with a combination of HER2 and PI3K inhibitors.

ErbB3 Ablation Impairs PI3K/Akt-Dependent Mammary Tumorigenesis

The ErbB receptor family member ErbB3 has been implicated in breast cancer growth, but it has yet to be determined whether its disruption is therapeutically valuable. In a mouse model of mammary carcinoma driven by the polyomavirus middle T (PyVmT) oncogene, the ErbB2 tyrosine kinase inhibitor lapatinib reduced the activation of ErbB3 and Akt as well as tumor cell growth. In this phosphatidylinositol-3 kinase (PI3K)-dependent tumor model, ErbB2 is part of a complex containing PyVmT, p85 (PI3K), and ErbB3, that is disrupted by treatment with lapatinib. Thus, full engagement of PI3K/Akt by ErbB2 in this oncogene-induced mouse tumor model may involve its ability to dimerize with and phosphorylate ErbB3, which itself directly binds PI3K. In this article, we report that ErbB3 is critical for PI3K/Akt-driven tumor formation triggered by the PyVmT oncogene. Tissue-specific, Cre-mediated deletion of ErbB3 reduced Akt phosphorylation, primary tumor growth, and pulmonary metastasis. Furthermore, EZN-3920, a chemically stabilized antisense oligonucleotide that targets the ErbB3 mRNA in vivo, produced similar effects while causing no toxicity in the mouse model. Our findings offer further preclinical evidence that ErbB3 ablation may be therapeutically effective in tumors where ErbB3 engages PI3K/Akt signaling.

Extracellular Signal-Regulated Kinase Is a Target of Cyclooxygenase-1-Peroxisome Proliferator-Activated Receptor-δ Signaling in Epithelial Ovarian Cancer

The underlying causes of epithelial ovarian cancer (EOC) are unclear, and treatment options for patients with advanced disease are limited. There is evidence that the use of nonsteroidal anti-inflammatory drugs is associated with decreased risk of developing EOC. Nonsteroidal anti-inflammatory drugs inhibit cyclooxygenase (COX)-1 and COX-2, which catalyze prostaglandin biosynthesis. We previously showed that mouse and human EOCs have increased levels of COX-1, but not COX-2, and a COX-1–selective inhibitor, SC-560, attenuates prostaglandin production and tumor growth. However, the downstream targets of COX-1 signaling in EOC are not yet known. To address this question, we evaluated peroxisome proliferator-activated receptor δ (PPARδ) expression and function in EOC. We found that EOC cells express high levels of PPARδ, and neutralizing PPARδ function reduces tumor growth in vivo . More interestingly, aspirin, a nonsteroidal anti-inflammatory drug that preferentially inhibits COX-1, compromises PPARδ function and cell growth by inhibiting extracellular signal-regulated kinases 1/2, members of the mitogen-activated protein kinase family. Our study, for the first time, shows that whereas PPARδ can be a target of COX-1, extracellular signal-regulated kinase is a potential target of PPARδ. The ability of aspirin to inhibit EOC growth in vivo is an exciting finding because of its low cost, lack of cardiovascular side effects, and availability. [Cancer Res 2007;67(11):5285–92]

Exon 9 and exon 20 mutations in PIK3CA confer resistance to HER2 inhibitors in HER2-overexpressing breast cancer cells.

Abstract #4054 The anti-tumor effect of HER2 antagonists in HER2-dependent breast cancer cells has been proposed to rely on inhibition of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Mutations in the p110α catalytic subunit of PI3K occur in up to 40% of breast cancers and activation of this pathway has been implicated in resistance to the HER2 antibody trastuzumab (T). PIK3CA mutations cluster in two regions in the helical (E542K, E545K; exon 9) and catalytic (H1047R; exon 20) domains. We studied the role of these mutants in resistance to HER2 inhibitors in breast cancer cells with HER2 amplification. Two lines with endogenous H1047R p110 (PI3K), SUM190 and HCC1954, and SKBR3, SUM225, and BT474 cells stably transduced with retroviral vectors encoding HA-tagged wild-type, E545K, or H1047R p110, were studied for their response to the HER2 tyrosine kinase inhibitor lapatinib ditosylate (L; GW-572016) and T. In monolayer and 3D cell proliferation assays, partial resistance to L and T was conferred by either mutation compared to WT p110. After prolonged L treatment, Ser473 phosphorylation of Akt recovered in all cells with endogenous or ectopic p110 mutants in spite of continued inhibition of Y1248 P-HER2 and Y1289 P-HER3 by L. Further, BT474 cells with either p110 mutant could be passaged in the continued presence of L. Immunoprecipitation of p85, the regulatory subunit of PI3K, showed that PI3K association with HER3 was abrogated by T and L. RNAi of HER3 in MCF10A/HER2/PI3KE545K and MCF10A/HER2/PI3KH1047R cells markedly but not completely inhibited growth suggesting that the PI3K mutants may still depend on HER3 for full activation. We hypothesized that in cells with mutant PI3K, the mutants coexist in a pool with WT enzyme, and that mutant and WT p110α are bound to p85 in variable proportions. Experiments to measure whether the ratio of mutant to WT p110 bound to p85, assayed by mass spectrometry, will increase in the PI3K-mutant cells with acquired resistance to L are in progress. To test the role of these mutants on resistance to anti-HER2 therapies in vivo, athymic mice bearing BT474 xenografts with ectopic WT, E545K, or H1047R p110 are undergoing treatment with L and T. Finally, we analyzed mutations of PIK3CA in a cohort of 40 patients with locally advanced HER2+ breast cancer treated with weekly single-agent T for 3 weeks, followed by T with docetaxel for a total of 12 weeks before surgery (Mohsin et al. JCO 23:2460, 2005). Sequential core biopsies at weeks 1 and 3 after initiation of T were taken. Eight of 40 tumors (20%) expressed mutant PI3K, 2 in exon 9 and 6 in exon 20. PIK3CA mutations did not correlate with change in Ki67 (p=0.97) or cleaved caspase 3 (p=0.51) in weeks 1 or 3 of treatment nor with pathologic complete response (p=0.21). These data suggest that, if mutant PI3K confers relative resistance to T, a shorter time to recurrence may be a more robust endpoint as the initial cellular or clinical response may not be a good indicator of this resistance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4054.