Stan Lipkowitz

Evaluation of Biologic End Points and Pharmacokinetics in Patients With Metastatic Breast Cancer After Treatment With Erlotinib, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor

PURPOSE To evaluate changes in epidermal growth factor receptor (EGFR) phosphorylation and its downstream signaling in tumor and surrogate tissue biopsies in patients with metastatic breast cancer treated with erlotinib, an EGFR tyrosine kinase inhibitor, and to assess relationships between biomarkers in tumor and normal tissues and between biomarkers and pharmacokinetics. PATIENTS AND METHODS Eighteen patients were treated orally with 150 mg/d of erlotinib. Ki67, EGFR, phosphorylated EGFR (pEGFR), phosphorylated mitogen-activated protein kinase (pMAPK), and phosphorylated AKT (pAKT) in 15 paired tumor, skin, and buccal mucosa biopsies (at baseline and after 1 month of therapy) were examined by immunohistochemistry and analyzed quantitatively. Pharmacokinetic sampling was also obtained. RESULTS The stratum corneum layer and Ki67 in keratinocytes of the epidermis in 15 paired skin biopsies significantly decreased after treatment (P = .0005 and P = .0003, respectively). No significant change in Ki67 was detected in 15 tumors, and no responses were observed. One was EGFR-positive and displayed heterogeneous expression of the receptor, and 14 were EGFR-negative. In the EGFR-positive tumor, pEGFR, pMAPK, and pAKT were reduced after treatment. Paradoxically, pEGFR was increased in EGFR-negative tumors post-treatment (P = .001). Although markers were reduced in surrogate and tumor tissues in the patient with EGFR-positive tumor, no apparent associations were observed in patients with EGFR-negative tumor. CONCLUSION Erlotinib has inhibitory biologic effects on normal surrogate tissues and on an EGFR-positive tumor. The lack of reduced tumor proliferation may be attributed to the heterogeneous expression of receptor in the EGFR-positive patient and absence of target in this cohort of heavily pretreated patients.

WW Domain HECT E3s Target Cbl RING Finger E3s for Proteasomal Degradation

Cbl proteins have RING finger-dependent ubiquitin ligase (E3) activity that is essential for down-regulation of tyrosine kinases. Here we establish that two WW domain HECT E3s, Nedd4 and Itch, bind Cbl proteins and target them for proteasomal degradation. This is dependent on the E3 activity of the HECT E3s but not on that of Cbl. Consistent with these observations, in cells expressing the epidermal growth factor receptor, Nedd4 reverses Cbl-b effects on receptor down-regulation, ubiquitylation, and proximal events in signaling. Cbl-b also targets active Src for degradation in cells, and Nedd4 similarly reverses Cbl-mediated Src degradation. These findings establish that RING finger E3s can be substrates, not only for autoubiquitylation but also for ubiquitylation by HECT E3s and suggest an additional level of regulation for Cbl substrates including protein-tyrosine kinases.

cbl-3: a new mammalian cbl family protein.

We have cloned a new human gene, cbl-3, which encodes a protein with marked homology to the cbl family of proteins. The predicted protein encoded by this gene retains the conserved phosphotyrosine binding domain (PTB) in the N-terminal and the zinc finger but is significantly shorter (MW 52.5 kDa) than the other mammalian cbl proteins. The protein lacks the extensive proline rich domain and leucine zipper seen in c-cbl and cbl-b and structurally most resembles the C. elegans and Drosophila cbl proteins. The gene is ubiquitously expressed with highest expression in the aerodigestive tract, prostate, adrenal gland, and salivary gland. The protein is phosphorylated and recruited to the EGFR upon EGF stimulation and inhibits EGF stimulated MAP kinase activation. In comparison to the other mammalian cbl proteins (e.g. cbl-b), cbl-3 interacts with a restricted range of proteins containing Src Homology 3 regions. An alternatively spliced form of the cbl-3 protein was also identified which deletes a critical region of the PTB domain and which does not interact with the EGFR nor inhibit EGF stimulated MAP kinase activation. These data demonstrate that cbl-3, a novel mammalian cbl protein, is a regulator of EGFR mediated signal transduction.

cbl -b inhibits epidermal growth factor receptor signaling

The role of cbl-b in signaling by the epidermal growth factor receptor (EGFR) was studied and compared with c-cbl. We demonstrate in vivo, that cbl-b, like c-cbl, is phosphorylated and recruited to the EGFR upon EGF stimulation and both cbl proteins can bind to the Grb2 adaptor protein. To investigate the functional role of cbl proteins in EGFR signaling, we transfected cbl-b or c-cbl into 32D cells overexpressing the EGFR (32D/EGFR). This cell line is absolutely dependent on exogenous IL-3 or EGF for sustained growth. 32D/EGFR cells overexpressing cbl-b showed markedly inhibited growth in EGF compared to c-cbl transfectants and vector controls. This growth inhibition by cbl-b was the result of a dramatic increase in the number of cells undergoing apoptosis. Consistent with this finding, cbl-b overexpression markedly decreased the amplitude and duration of AKT activation upon EGF stimulation compared to either vector controls or c-cbl overexpressing cells. In addition, the duration of EGF mediated MAP kinase and Jun kinase activation in cells overexpressing cbl-b is shortened. These data demonstrate that cbl-b inhibits EGF-induced cell growth and that cbl-b and c-cbl have distinct roles in EGF mediated signaling.

Inhibition of NF-κB Activity Enhances TRAIL Mediated Apoptosis in Breast Cancer Cell Lines

Most breast cancer cell lines are resistant to TNF-related apoptosis inducing ligand (TRAIL) induced apoptosis. In sensitive breast cancer cell lines TRAIL rapidly induces the cleavage and activation of caspases leading to the subsequent cleavage of downstream caspase substrates. In contrast, there is no caspase activation in the resistant cell lines. The transcription factor NF-κB can inhibit apoptosis induced by a variety of stimuli including activation of death receptors. We investigated whether NF-κB contributes to the resistance of breast cancer cells to TRAIL induced apoptosis. All of the resistant breast cancer cell lines expressed NF-κB and had detectable NF-κB activity in nuclear extracts prior to treatment with TRAIL. Upon TRAIL treatment, a significant increase in NF-κB activity was seen in most of the cell lines. To directly test if NF-κB activity contributes to the resistance of these cell lines to TRAIL, we transiently transfected the resistant cell lines with an inhibitor of NF-κB (IκBΔN) and measured TRAIL induced apoptosis in control and transfected cells. All of the resistant cell lines tested showed an increase in TRAIL induced apoptosis when transfected with the IκBΔN. These results demonstrate that TRAIL resistant breast cancer cells fail to rapidly activate the apoptotic machinery but they do activate NF-κB. Inhibition of NF-κB activity increases the sensitivity to TRAIL mediated apoptosis in resistant cells. These results suggest that agents which inhibit NF-κB should increase the clinical efficacy of TRAIL in breast cancer cells.

Cbl-b interacts with ubiquitinated proteins ; differential functions of the UBA domains of c-Cbl and Cbl-b

Cbl proteins are ubiquitin protein ligases, which ubiquitinate activated tyrosine kinases and target them for degradation. Both c-Cbl and Cbl-b have an ubiquitin associated (UBA) domain at their C-terminal end. We observed that high molecular weight ubiquitinated proteins constitutively coimmunoprecipitated with transfected and endogenous Cbl-b, but not c-Cbl. The binding site for these ubiquitinated proteins was mapped to the UBA domain of Cbl-b (UBAb). GST-fusion proteins containing the UBAb interacted with ubiquitinated proteins and polyubiquitin chains in vitro, whereas those containing the UBA domain of c-Cbl (UBAc) did not. The UBAb had a much greater affinity for polyubiquitin chains than for monoubiquitin. Analysis of the UBAb and UBAc demonstrate that the affinity for ubiquitin is determined by multiple amino-acid differences between the two domains. Overexpression of the UBAb, but not overexpression of the UBAc, inhibited a variety of ubiquitin-mediated processes such as degradation of ubiquitinated proteins (i.e. EGFR, Mdm-2, and Siah-1). This in vivo result is consistent with the differences in ubiquitin binding observed in vitro between the UBAb and UBAc. This difference in ubiquitin-binding may reflect distinct regulatory functions of c-Cbl and Cbl-b.

Apoptosis is associated with cleavage of a 5 kDa fragment from RB which mimics dephosphorylation and modulates E2F binding.

Dephosphorylation of the RB protein has been reported to be associated with apoptosis. In contrast, we show that treatment of HL60 cells with etoposide or cytosine arabinoside or treatment of breast epithelial cells with α-FAS is associated with the cleavage of a 5 kDa fragment from the C-terminus of RB, resulting in a truncated product that we have designated as p100cl. This cleavage event coincides with the activation of cysteine proteases at the onset of apoptosis, is blocked by the addition of iodoacetamide to cells prior to the onset of apoptosis, and results in the expression of faster migrating protein species which can mimic dephosphorylated RB. The free 5 kDa fragment is detected only during apoptosis, predicts a cleavage site that we have mapped to a unique CPP32-like recognition sequence which is present at the C-terminus of all reported RB homologues, and results in a truncated RB protein with enhanced E2F binding affinity. While the causality for this cleavage event in the apoptotic process is still under investigation, our findings suggest distinct post-translational pathways for the RB product between cells examined during growth arrest (p105 hypophosphorylated RB) or apoptosis (p100cl).

SETA is a multifunctional adapter protein with three SH3 domains that binds Grb2, Cbl, and the novel SB1 proteins

Expression of the src homology 3 (SH3)-encoding, expressed in tumorigenic astrocytes (SETA) gene is associated with astrocyte transformation in culture and tumors in the adult brain. SETA binds to the apoptosis regulator apoptosis-linked gene 2 (ALG-2) interacting protein 1 (AIP1), and modulates apoptosis in astrocytes. The predicted protein structure of SETA revealed two SH3 domains, while related proteins were reported to have three. Here we report the identification of an additional SH3 domain N-terminal to the previously identified SETA sequence. Yeast two-hybrid screening of a p53(-/-) astrocyte cDNA library with this SH3 domain identified a novel gene, SETA binding protein 1 (SB1), with 55% amino acid identity to the renal tumor antigen, NY-REN-45. In vitro confrontation and co-immunoprecipitation experiments confirmed the binding of SB1 to SETA. Evidence that SETA binds to the CD2 protein, the proto-oncogene c-Cbl, and the signal transduction molecule Grb2, and can dimerize via its C-terminal coiled coil (CC) domain is also presented.

Randomized Comparison of Group Versus Individual Genetic Education and Counseling for Familial Breast and/or Ovarian Cancer

PURPOSE An efficient approach to education and counseling before BRCA1 and BRCA2 mutation testing is necessary for effective utilization of testing in the community. Education and counseling, when delivered individually, are limited by a shortage of trained health care providers as well as by financial and time constraints. The purpose of this study was to determine whether pretest education and counseling for breast cancer genetics in a group setting is equivalent to that provided on an individual basis. PATIENTS AND METHODS One hundred forty-two patients at high risk for harboring a BRCA mutation were randomly assigned to group or individual education and counseling sessions. Group education was followed by brief individual counseling. Knowledge and Impact of Events Scales (IES) were administered at baseline and after education and counseling and at 1 week and 3, 6, and 12 months. Satisfaction with education and counseling was measured at completion of the session. Preferred method of education and counseling was solicited at 3 months. RESULTS There was no difference in knowledge or IES scores between groups. When stratified by genetic test results, knowledge scores showed no difference. Regardless of group, post-test IES scores in patients with positive results were higher than patients with negative or uninformative results but returned to baseline by 12 months. Participants were equally satisfied with either method they were assigned. Significantly more time was spent per patient in individual sessions (1.25 hours) than in group education (0.74 hours). CONCLUSION Our data suggest that group education and counseling may confer similar benefits compared with traditional individual sessions. Additional investigation of this approach in larger numbers of patients is warranted.

Cbl-3-deficient mice exhibit normal epithelial development

ABSTRACT Cbl family proteins are evolutionarily conserved ubiquitin ligases that negatively regulate signaling from tyrosine kinase-coupled receptors. The mammalian cbl family consists of c-Cbl, Cbl-b, and the recently cloned Cbl-3 (also known as Cbl-c). In this study, we describe the detailed expression pattern of murine Cbl-3 and report the generation and characterization of Cbl-3-deficient mice. Cbl-3 exhibits an expression pattern distinct from those of c-Cbl and Cbl-b, with high levels of Cbl-3 expression in epithelial cells of the gastrointestinal tract and epidermis, as well as the respiratory, urinary, and reproductive systems. Cbl-3 expression was not detected in nonepithelial cells, but within epithelial tissues, the levels of Cbl-3 expression varied from undetectable in the alveoli of the lungs to very strong in the cecum and colon. Despite this restricted expression pattern, Cbl-3-deficient mice were viable, healthy, and fertile and displayed no histological abnormalities up to 18 months of age. Proliferation of epithelial cells in the epidermises and gastrointestinal tracts was unaffected by the loss of Cbl-3. Moreover, Cbl-3 was not required for attenuation of epidermal growth factor-stimulated Erk activation in primary keratinocytes. Thus, Cbl-3 is dispensable for normal epithelial development and function.