J. Arribas

NVP-BEZ235, a Dual PI3K/mTOR Inhibitor, Prevents PI3K Signaling and Inhibits the Growth of Cancer Cells with Activating PI3K Mutations

Phosphatidylinositol-3-kinase (PI3K) pathway deregulation is a common event in human cancer, either through inactivation of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 or activating mutations of p110-alpha. These hotspot mutations result in oncogenic activity of the enzyme and contribute to therapeutic resistance to the anti-HER2 antibody trastuzumab. The PI3K pathway is, therefore, an attractive target for cancer therapy. We have studied NVP-BEZ235, a dual inhibitor of the PI3K and the downstream mammalian target of rapamycin (mTOR). NVP-BEZ235 inhibited the activation of the downstream effectors Akt, S6 ribosomal protein, and 4EBP1 in breast cancer cells. The antiproliferative activity of NVP-BEZ235 was superior to the allosteric selective mTOR complex inhibitor everolimus in a panel of 21 cancer cell lines of different origin and mutation status. The described Akt activation due to mTOR inhibition was prevented by higher doses of NVP-BEZ235. NVP-BEZ235 reversed the hyperactivation of the PI3K/mTOR pathway caused by the oncogenic mutations of p110-alpha, E545K, and H1047R, and inhibited the proliferation of HER2-amplified BT474 cells exogenously expressing these mutations that render them resistant to trastuzumab. In trastuzumab-resistant BT474 H1047R breast cancer xenografts, NVP-BEZ235 inhibited PI3K signaling and had potent antitumor activity. In treated animals, there was complete inhibition of PI3K signaling in the skin at pharmacologically active doses, suggesting that skin may serve as surrogate tissue for pharmacodynamic studies. In summary, NVP-BEZ235 inhibits the PI3K/mTOR axis and results in antiproliferative and antitumoral activity in cancer cells with both wild-type and mutated p110-alpha.

PI3K inhibition results in enhanced HER signaling and acquired ERK dependency in HER2-overexpressing breast cancer.

There is a strong rationale to therapeutically target the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway in breast cancer since it is highly deregulated in this disease and it also mediates resistance to anti-HER2 therapies. However, initial studies with rapalogs, allosteric inhibitors of mTORC1, have resulted in limited clinical efficacy probably due to the release of a negative regulatory feedback loop that triggers AKT and ERK signaling. Since activation of AKT occurs via PI3K, we decided to explore whether PI3K inhibitors prevent the activation of these compensatory pathways. Using HER2-overexpressing breast cancer cells as a model, we observed that PI3K inhibitors abolished AKT activation. However, PI3K inhibition resulted in a compensatory activation of the ERK signaling pathway. This enhanced ERK signaling occurred as a result of activation of HER family receptors as evidenced by induction of HER receptors dimerization and phosphorylation, increased expression of HER3 and binding of adaptor molecules to HER2 and HER3. The activation of ERK was prevented with either MEK inhibitors or anti-HER2 monoclonal antibodies and tyrosine kinase inhibitors. Combined administration of PI3K inhibitors with either HER2 or MEK inhibitors resulted in decreased proliferation, enhanced cell death and superior anti-tumor activity compared with single agent PI3K inhibitors. Our findings indicate that PI3K inhibition in HER2-overexpressing breast cancer activates a new compensatory pathway that results in ERK dependency. Combined anti-MEK or anti-HER2 therapy with PI3K inhibitors may be required in order to achieve optimal efficacy in HER2-overexpressing breast cancer. This approach warrants clinical evaluation.

Lapatinib, a HER2 tyrosine kinase inhibitor, induces stabilization and accumulation of HER2 and potentiates trastuzumab-dependent cell cytotoxicity

Lapatinib is a human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor (TKI) that has clinical activity in HER2-amplified breast cancer. In vitro studies have shown that lapatinib enhances the effects of the monoclonal antibody trastuzumab suggesting partially non-overlapping mechanisms of action. To dissect these mechanisms, we have studied the effects of lapatinib and trastuzumab on receptor expression and receptor signaling and have identified a new potential mechanism for the enhanced antitumor activity of the combination. Lapatinib, given alone or in combination with trastuzumab to HER2-overexpressing breast cancer cells SKBR3 and MCF7-HER2, inhibited HER2 phosphorylation, prevented receptor ubiquitination and resulted in a marked accumulation of inactive receptors at the cell surface. By contrast, trastuzumab alone caused enhanced HER2 phosphorylation, ubiquitination and degradation of the receptor. By immunoprecipitation and computational protein modeling techniques we have shown that the lapatinib-induced HER2 accumulation at the cell surface also results in the stabilization of inactive HER2 homo- (HER2/HER2) and hetero- (HER2/EGFR and HER2/HER3) dimers. Lapatinib-induced accumulation of HER2 and trastuzumab-mediated downregulation of HER2 was also observed in vivo, where the combination of the two agents triggered complete tumor remissions in all cases after 10 days of treatment. Accumulation of HER2 at the cell surface by lapatinib enhanced immune-mediated trastuzumab-dependent cytotoxicity. We propose that this is a novel mechanism of action of the combination that may be clinically relevant and exploitable in the therapy of patients with HER2-positive tumors.

Clinical Benefit of Lapatinib-Based Therapy in Patients with Human Epidermal Growth Factor Receptor 2–Positive Breast Tumors Coexpressing the Truncated p95HER2 Receptor

Purpose: A subgroup of human epidermal growth factor receptor 2 (HER2)–overexpressing breast tumors coexpresses p95HER2, a truncated HER2 receptor that retains a highly functional HER2 kinase domain but lacks the extracellular domain and results in intrinsic trastuzumab resistance. We hypothesized that lapatinib, a HER2 tyrosine kinase inhibitor, would be active in these tumors. We have studied the correlation between p95HER2 expression and response to lapatinib, both in preclinical models and in the clinical setting. Experimental Design: Two different p95HER2 animal models were used for preclinical studies. Expression of p95HER2 was analyzed in HER2-overexpressing breast primary tumors from a first-line lapatinib monotherapy study (EGF20009) and a second-line lapatinib in combination with capecitabine study (EGF100151). p95HER2 expression was correlated with overall response rate (complete + partial response), clinical benefit rate (complete response + partial response + stable disease ≥24 wk), and progression-free survival using logistic regression and Cox proportional hazard models. Results: Lapatinib inhibited tumor growth and the HER2 downstream signaling of p95HER2-expressing tumors. A total of 68 and 156 tumors from studies EGF20009 and EGF100151 were evaluable, respectively, for p95HER2 detection. The percentage of p95HER2-positive patients was 20.5% in the EGF20009 study and 28.5% in the EGF100151 study. In both studies, there was no statistically significant difference in progression-free survival, clinical benefit rate, and overall response rate between p95HER2-positive and p95HER2-negative tumors. Conclusions: Lapatinib as a monotherapy or in combination with capecitabine seems to be equally effective in patients with p95HER2-positive and p95HER2-negative HER2-positive breast tumors. Clin Cancer Res; 16(9); 2688–95. ©2010 AACR.

Proteomic Identification of Desmoglein 2 and Activated Leukocyte Cell Adhesion Molecule as Substrates of ADAM17 and ADAM10 by Difference Gel Electrophoresis

ABSTRACT In contrast with the early view of metalloproteases as simple extracellular matrix-degrading entities, recent findings show that they are highly specific modulators of different signaling pathways involved, positively or negatively, in tumor development. Thus, before considering a given metalloprotease a therapeutic target, it seems advisable to characterize its function by identifying its repertoire of substrates. Here, we present a proteomic approach to identify ADAM17 substrates by difference gel electrophoresis. We found that the shedding of the extracellular domain of the transferrin receptor and those of two cell-cell adhesion molecules, activated leukocyte cell adhesion molecule (ALCAM) and desmoglein 2 (Dsg-2), is increased in cells overexpressing ADAM17. Genetic evidence shows that while ADAM17 is responsible for the shedding of ALCAM, both ADAM17 and ADAM10 can act on Dsg-2. Activation of the epidermal growth factor receptor leads to the upregulation of the shedding of Dsg-2 and to the concomitant upregulation of ADAM17, but not ADAM10, supporting the ability of overexpressed ADAM17 to shed Dsg-2. These results unveil a role of ADAM10 and ADAM17 in the shedding of cell-cell adhesion molecules. Since loss of cell adhesion is an early event in tumor development, these results suggest that ADAM17 is a useful target in anticancer therapy.

Inhibitors of HSP90 block p95-HER2 signaling in Trastuzumab-resistant tumors and suppress their growth.

The anti-HER2 antibody Trastuzumab (Herceptin) has been proven to be effective in the treatment of HER2-overexpressing breast cancer; resistance, however, invariably emerges in metastatic tumors. The expression of p95-HER2, a form of HER2 with a truncated extracellular domain that lacks the Trastuzumab binding epitope, has been implicated as a mechanism of resistance to the antibody. We utilized an in vivo tumor model that overexpresses p95-HER2 and showed it to be resistant to the signaling and antitumor effects of Trastuzumab. We find that both full-length and p95-HER2 interact with the HSP90 chaperone protein and are degraded in tumor cells exposed to HSP90 inhibitors in tissue culture and in vivo. Loss of expression of p95-HER2 is accompanied by downregulation of the phosphoinositide-3 kinase/AKT and extracellular signal-regulated kinase signaling pathways and inhibition of cell proliferation. Chronic administration of HSP90 inhibitors in vivo results in sustained loss of HER2 and p95-HER2 expression and inhibition of AKT activation, together with induction of apoptosis and complete inhibition of tumor growth in Trastuzumab-resistant, p95-HER2-overexpressing models. Thus, p95-HER2 is an HSP90 client protein, the expression and function of which can be effectively suppressed in vivo by HSP90 inhibitors. HSP90 inhibition is therefore a potentially effective therapeutic strategy for p95-HER2-mediated Trastuzumab-resistant breast cancer.

Interrogating open issues in cancer precision medicine with patient-derived xenografts

Patient-derived xenografts (PDXs) have emerged as an important platform to elucidate new treatments and biomarkers in oncology. PDX models are used to address clinically relevant questions, including the contribution of tumour heterogeneity to therapeutic responsiveness, the patterns of cancer evolutionary dynamics during tumour progression and under drug pressure, and the mechanisms of resistance to treatment. The ability of PDX models to predict clinical outcomes is being improved through mouse humanization strategies and the implementation of co-clinical trials, within which patients and PDXs reciprocally inform therapeutic decisions. This Opinion article discusses aspects of PDX modelling that are relevant to these questions and highlights the merits of shared PDX resources to advance cancer medicine from the perspective of EurOPDX, an international initiative devoted to PDX-based research.

Microsatellite instability due to hMLH1 deficiency is associated with increased cytotoxicity to irinotecan in human colorectal cancer cell lines

Around 15% of colorectal cancers (CRCs) show microsatellite instability (MSI) due to dysfunction of the mismatch repair system (MMR). As a consequence of this, MSI tumours tend to accumulate errors in mononucleotide repeats as those in genes implicated in repairing double-strand breaks (DSBs). Previous studies have shown that irinotecan (CPT-11), a chemotherapy agent inducing DSB, is more active in MSI than in microsatellite stable (MSS) CRC. The purpose of this study was to compare the sensitivity to CPT-11 in a series of CRC cell lines with either proficient or deficient MMR and to assess the mutational status of two DSB repair genes, MRE11 and RAD50, in these cell lines. hMLH1-deficient cell lines due to either epigenetic silencing or mutation showed very similar IC50 and were four- to nine-fold more sensitive to CPT-11 than the MSS line. Cell lines harbouring mutations in both MRE11 and RAD50 were most sensitive to CPT-11. We conclude that MSI cell lines display higher sensitivity to CPT-11 than MSS cells. Mutation of MRE11 and RAD50 could account for this difference in response to CPT-11. Future clinical trials tailoring chemotherapy regimens based on microsatellite status are warranted.

RSK3/4 mediate resistance to PI3K pathway inhibitors in breast cancer

The PI3K signaling pathway regulates diverse cellular processes, including proliferation, survival, and metabolism, and is aberrantly activated in human cancer. As such, numerous compounds targeting the PI3K pathway are currently being clinically evaluated for the treatment of cancer, and several have shown some early indications of efficacy in breast cancer. However, resistance against these agents, both de novo and acquired, may ultimately limit the efficacy of these compounds. Here, we have taken a systematic functional approach to uncovering potential mechanisms of resistance to PI3K inhibitors and have identified several genes whose expression promotes survival under conditions of PI3K/mammalian target of rapamycin (PI3K/mTOR) blockade, including the ribosomal S6 kinases RPS6KA2 (RSK3) and RPS6KA6 (RSK4). We demonstrate that overexpression of RSK3 or RSK4 supports proliferation upon PI3K inhibition both in vitro and in vivo, in part through the attenuation of the apoptotic response and upregulation of protein translation. Notably, the addition of MEK- or RSK-specific inhibitors can overcome these resistance phenotypes, both in breast cancer cell lines and patient-derived xenograft models with elevated levels of RSK activity. These observations provide a strong rationale for the combined use of RSK and PI3K pathway inhibitors to elicit favorable responses in breast cancer patients with activated RSK.

SFRPs act as negative modulators of ADAM10 to regulate retinal neurogenesis

It is well established that retinal neurogenesis in mouse embryos requires the activation of Notch signaling, but is independent of the Wnt signaling pathway. We found that genetic inactivation of Sfrp1 and Sfrp2, two postulated Wnt antagonists, perturbs retinal neurogenesis. In retinas from Sfrp1−/−; Sfrp2−/− embryos, Notch signaling was transiently upregulated because Sfrps bind ADAM10 metalloprotease and downregulate its activity, an important step in Notch activation. The proteolysis of other ADAM10 substrates, including APP, was consistently altered in Sfrp mutants, whereas pharmacological inhibition of ADAM10 partially rescued the Sfrp1−/−; Sfrp2−/− retinal phenotype. Conversely, ectopic Sfrp1 expression in the Drosophila wing imaginal disc prevented the expression of Notch targets, and this was restored by the coexpression of Kuzbanian, the Drosophila ADAM10 homolog. Together, these data indicate that Sfrps inhibit the ADAM10 metalloprotease, which might have important implications in pathological events, including cancer and Alzheimers disease.