Heidi Lane

ERBB receptors and cancer: the complexity of targeted inhibitors.

ERBB receptor tyrosine kinases have important roles in human cancer. In particular, the expression or activation of epidermal growth factor receptor and ERBB2 are altered in many epithelial tumours, and clinical studies indicate that they have important roles in tumour aetiology and progression. Accordingly, these receptors have been intensely studied to understand their importance in cancer biology and as therapeutic targets, and many ERBB inhibitors are now used in the clinic. We will discuss the significance of these receptors as clinical targets, in particular the molecular mechanisms underlying response.

The ErbB signaling network: receptor heterodimerization in development and cancer

Cells are continuously exposed to diverse stimuli ranging from soluble endocrine and paracrine factors, to signaling molecules on neighboring cells. It is of great importance that these extracellular signals are correctly interpreted by the cell, in order to achieve an appropriate developmental or proliferative response. Receptors of the tyrosine kinase family play pivotal roles in this process. By binding to specific peptide ligands they are able to integrate these external stimuli with internal signal transduction pathways, contributing in this fashion to the ability of the cell to respond correctly to its environment. In this review, we will concentrate on the role of ErbB receptors as normal signal transducers and their contribution to the process of malignant transformation during tumor development. ErbB proteins belong to subclass I of the superfamily of receptor tyrosine kinases (RTKs). There are four members of the ErbB family: epidermal growth factor (EGF) receptor (also termed ErbB1/HER1), ErbB2/Neu/HER2, ErbB3/HER3 and ErbB4/HER4. We will refer to them, henceforth, as the ErbB receptors. All family members have in common an extracellular ligand‐binding domain, a single membrane‐spanning region and a cytoplasmic protein tyrosine kinase domain. A family of ligands, the EGF‐related peptide growth factors, bind the extracellular domain of ErbB receptors leading to the formation of both homo‐ and heterodimers. Dimerization consequently stimulates the intrinsic tyrosine kinase activity of the receptors and triggers autophosphorylation of specific tyrosine residues within the cytoplasmic domain. These phosphorylated residues serve as docking sites for signaling molecules involved in the regulation of intracellular signaling cascades. Ultimately, downstream effects on gene expression determine the biological response to receptor activation. ErbB receptors are expressed in a variety of tissues of epithelial, mesenchymal and neuronal origin, where they play fundamental roles in development, proliferation and differentiation. Moreover, deregulated expression of ErbB receptors, in particular ErbB1 and ErbB2, has …

mTOR Inhibition Induces Upstream Receptor Tyrosine Kinase Signaling and Activates Akt

Stimulation of the insulin and insulin-like growth factor I (IGF-I) receptor activates the phosphoinositide-3-kinase/Akt/mTOR pathway causing pleiotropic cellular effects including an mTOR-dependent loss in insulin receptor substrate-1 expression leading to feedback down-regulation of signaling through the pathway. In model systems, tumors exhibiting mutational activation of phosphoinositide-3-kinase/Akt kinase, a common event in cancers, are hypersensitive to mTOR inhibitors, including rapamycin. Despite the activity in model systems, in patients, mTOR inhibitors exhibit more modest antitumor activity. We now show that mTOR inhibition induces insulin receptor substrate-1 expression and abrogates feedback inhibition of the pathway, resulting in Akt activation both in cancer cell lines and in patient tumors treated with the rapamycin derivative, RAD001. IGF-I receptor inhibition prevents rapamycin-induced Akt activation and sensitizes tumor cells to inhibition of mTOR. In contrast, IGF-I reverses the antiproliferative effects of rapamycin in serum-free medium. The data suggest that feedback down-regulation of receptor tyrosine kinase signaling is a frequent event in tumor cells with constitutive mTOR activation. Reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy that ablates mTOR function and prevents Akt activation may have improved antitumor activity.

mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways.

Loss of PTEN function leads to activation of phosphoinositide 3-kinase (PI3K) signaling and Akt. Clinical trials are now testing whether mammalian target of rapamycin (mTOR) inhibition is useful in treating PTEN-null cancers. Here, we report that mTOR inhibition induced apoptosis of epithelial cells and the complete reversal of a neoplastic phenotype in the prostate of mice expressing human AKT1 in the ventral prostate. Induction of cell death required the mitochondrial pathway, as prostate-specific coexpression of BCL2 blocked apoptosis. Thus, there is an mTOR-dependent survival signal required downstream of Akt. Bcl2 expression, however, only partially restored intraluminal cell growth in the setting of mTOR inhibition. Expression profiling showed that Hif-1α targets, including genes encoding most glycolytic enzymes, constituted the dominant transcriptional response to AKT activation and mTOR inhibition. These data suggest that the expansion of AKT-driven prostate epithelial cells requires mTOR-dependent survival signaling and activation of HIF-1α, and that clinical resistance to mTOR inhibitors may emerge through BCL2 expression and/or upregulation of HIF-1α activity.

Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo

We have isolated a human homolog of Xenopus Eg5, a kinesin-related motor protein implicated in the assembly and dynamics of the mitotic spindle. We report that microinjection of antibodies against human Eg5 (HsEg5) blocks centrosome migration and causes HeLa cells to arrest in mitosis with monoastral microtubule arrays. Furthermore, an evolutionarily conserved cdc2 phosphorylation site (Thr-927) in HsEg5 is phosphorylated specifically during mitosis in HeLa cells and by p34cdc2/cyclin B in vitro. Mutation of Thr-927 to nonphosphorylatable residues prevents HsEg5 from binding to centrosomes, indicating that phosphorylation controls the association of this motor with the spindle apparatus. These results indicate that HsEg5 is required for establishing a bipolar spindle and that p34cdc2 protein kinase directly regulates its localization.

Phase II Randomized Study of Neoadjuvant Everolimus Plus Letrozole Compared With Placebo Plus Letrozole in Patients With Estrogen Receptor-Positive Breast Cancer

PURPOSE Cross-talk between the estrogen receptor (ER) and the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways is a mechanism of resistance to endocrine therapy, and blockade of both pathways enhances antitumor activity in preclinical models. This study explored whether sensitivity to letrozole was enhanced with the oral mTOR inhibitor, everolimus (RAD001). PATIENTS AND METHODS Two hundred seventy postmenopausal women with operable ER-positive breast cancer were randomly assigned to receive 4 months of neoadjuvant treatment with letrozole (2.5 mg/day) and either everolimus (10 mg/day) or placebo. The primary end point was clinical response by palpation. Mandatory biopsies were obtained at baseline and after 2 weeks of treatment (ie, day 15). Samples were assessed for PI3K mutation status (PIK3CA) and for pharmacodynamic changes of Ki67, phospho-S6, cyclin D1, and progesterone receptor (PgR) by immunohistochemistry. RESULTS Response rate by clinical palpation in the everolimus arm was higher than that with letrozole alone (ie, placebo; 68.1% v 59.1%), which was statistically significant at the preplanned, one-sided, alpha = 0.1 level (P = .062). Marked reductions in progesterone receptor and cyclin D1 expression occurred in both treatment arms, and dramatic downregulation of phospho-S6 occurred only in the everolimus arm. An antiproliferative response, as defined by a reduction in Ki67 expression to natural logarithm of percentage positive Ki67 of less than 1 at day 15, occurred in 52 (57%) of 91 patients in the everolimus arm and in 25 (30%) of 82 patients in the placebo arm (P < .01). The safety profile was consistent with historical results of everolimus monotherapy; grades 3 to 4 adverse events occurred in 22.6% of patients who received everolimus and in 3.8% of patients who received placebo. CONCLUSION Everolimus significantly increased letrozole efficacy in neoadjuvant therapy of patients with ER-positive breast cancer.

Dose- and Schedule-Dependent Inhibition of the Mammalian Target of Rapamycin Pathway With Everolimus: A Phase I Tumor Pharmacodynamic Study in Patients With Advanced Solid Tumors

PURPOSE Everolimus is a selective mammalian target of rapamycin (mTOR) inhibitor with promising anticancer activity. In order to identify a rationally based dose and schedule for cancer treatment, we have conducted a tumor pharmacodynamic phase I study in patients with advanced solid tumors. PATIENTS AND METHODS Fifty-five patients were treated with everolimus in cohorts of 20, 50, and 70 mg weekly or 5 and 10 mg daily. Dose escalation depended on dose limiting toxicity (DLT) rate during the first 4-week period. Pre- and on-treatment steady-state tumor and skin biopsies were evaluated for total and phosphorylated (p) protein S6 kinase 1, eukaryotic initiation factor 4E (elF-4E) binding protein 1 (4E-BP1), eukaryotic initiation factor 4G (eIF-4G), AKT, and Ki-67 expression. Plasma trough levels of everolimus were determined on a weekly basis before dosing during the first 4 weeks. RESULTS We observed a dose- and schedule-dependent inhibition of the mTOR pathway with a near complete inhibition of pS6 and peIF-4G at 10 mg/d and >or= 50 mg/wk. In addition, pAKT was upregulated in 50% of the treated tumors. In the daily schedule, there was a correlation between everolimus plasma trough concentrations and inhibition of peIF4G and p4E-BP1. There was good concordance of mTOR pathway inhibition between skin and tumor. Clinical benefit was observed in four patients including one patient with advanced colorectal cancer achieving a partial response. DLTs occurred in five patients: one patient at 10 mg/d (grade 3 stomatitis) and four patients at 70 mg/wk (two with grade 3 stomatitis, one with grade 3 neutropenia, and one with grade 3 hyperglycemia). CONCLUSION Everolimus achieved mTOR signaling inhibition at doses below the DLT. A dosage of 10 mg/d or 50 mg/wk is recommended for further development.

The mTOR Inhibitor RAD001 Sensitizes Tumor Cells to DNA-Damaged Induced Apoptosis through Inhibition of p21 Translation

Although DNA damaging agents have revolutionized chemotherapy against solid tumors, a narrow therapeutic window combined with severe side effects has limited their broader use. Here we show that RAD001 (everolimus), a rapamycin derivative, dramatically enhances cisplatin-induced apoptosis in wild-type p53, but not mutant p53 tumor cells. The use of isogenic tumor cell lines expressing either wild-type mTOR cDNA or a mutant that does not bind RAD001 demonstrates that the effects of RAD001 are through inhibition of mTOR function. We further show that RAD001 sensitizes cells to cisplatin by inhibiting p53-induced p21 expression. Unexpectedly, this effect is attributed to a small but significant inhibition of p21 translation combined with its short half-life. These findings provide the molecular rationale for combining DNA damaging agents with RAD001, showing that a general effect on a major anabolic process may dramatically enhance the efficacy of an established drug protocol in the treatment of cancer patients with solid tumors.

Phase I Pharmacokinetic and Pharmacodynamic Study of the Oral Mammalian Target of Rapamycin Inhibitor Everolimus in Patients With Advanced Solid Tumors

PURPOSE To identify the optimal regimen and dosage of the oral mammalian target of rapamycin inhibitor everolimus (RAD001). METHODS We performed a dose-escalation study in advanced cancer patients administering oral everolimus 5 to 30 mg/wk, with pharmacokinetic (PK) and pharmacodynamic (PD) studies. PD data prompted investigation of 50 and 70 mg weekly and daily dosing at 5 and 10 mg. RESULTS Ninety-two patients were treated. Dose-limiting toxicity was seen in one patient each at 50 mg/wk (stomatitis and fatigue) and 10 mg/d (hyperglycemia); hence, the maximum-tolerated dose was not reached. S6 kinase 1 activity in peripheral-blood mononuclear cells was inhibited for at least 7 days at doses >or= 20 mg/wk. Area under the curve increased proportional to dose, but maximum serum concentration increased less than proportionally at doses >or= 20 mg/wk. Terminal half-life was 30 hours (range, 26 to 38 hours). Partial responses were observed in four patients, and 12 patients remained progression free for >or= 6 months, including five of 10 patients with renal cell carcinoma. CONCLUSION Everolimus was satisfactorily tolerated at dosages up to 70 mg/wk and 10 mg/d with predictable PK. Antitumor activity and PD in tumors require further clinical investigation. Doses of 20 mg/wk and 5 mg/d are recommended as appropriate starting doses for these studies.

Response of a Neuronal Model of Tuberous Sclerosis to Mammalian Target of Rapamycin (mTOR) Inhibitors: Effects on mTORC1 and Akt Signaling Lead to Improved Survival and Function

Tuberous sclerosis (TSC) is a hamartoma syndrome attributable to mutations in either TSC1 or TSC2 in which brain involvement causes epilepsy, mental retardation, and autism. We have reported recently (Meikle et al., 2007) a mouse neuronal model of TSC in which Tsc1 is ablated in most neurons during cortical development. We have tested rapamycin and RAD001 [40-O-(2-hydroxyethyl)-rapamycin], both mammalian target of rapamycin mTORC1 inhibitors, as potential therapeutic agents in this model. Median survival is improved from 33 d to more than 100 d; behavior, phenotype, and weight gain are all also markedly improved. There is brain penetration of both drugs, with accumulation over time with repetitive treatment, and effective reduction of levels of phospho-S6, a downstream target of mTORC1. In addition, there is restoration of phospho-Akt and phospho-glycogen synthase kinase 3 levels in the treated mice, consistent with restoration of Akt function. Neurofilament abnormalities, myelination, and cell enlargement are all improved by the treatment. However, dysplastic neuronal features persist, and there are only modest changes in dendritic spine density and length. Strikingly, mice treated with rapamycin or RAD001 for 23 d only (postnatal days 7–30) displayed a persistent improvement in phenotype, with median survival of 78 d. In summary, rapamycin/RAD001 are highly effective therapies for this neuronal model of TSC, with benefit apparently attributable to effects on mTORC1 and Akt signaling and, consequently, cell size and myelination. Although caution is appropriate, the results suggest the possibility that rapamycin/RAD001 may have benefit in the treatment of TSC brain disease, including infantile spasms.