BRCA1 protein levels and PIK3CA mutations as predictive biomarkers for response to neoadjuvant chemotherapy in locally advanced breast cancer: An exploratory analysis.

BRCA1 overexpression and phosphoinositide 3-kinase (PIK3CA) pathway activation are involved in the resistance to DNA damaging agents. Thus, we hypothesized that BRCA1 protein expression and activating PIK3CA mutations are potential tumor biomarkers for the chemotherapeutic response to doxorubicin/cyclophosphamide plus docetaxel in locally advanced breast cancer. Informed consent was obtained and clinical, pathological and response data were collected. BRCA1 protein expression levels were assessed by immunohistochemistry of the archived tissue by two independent pathologists. The PIK3CA mutation status was assessed by nested PCR amplification and DNA sequencing. BRCA1 protein levels and the PIK3CA mutation status were correlated with pathological complete response and a partial response or better using the Chi-square test, Fisher's exact test and logistic regression. Of the 136 eligible participants, 59 samples could be analyzed. There was a trend of relatively low levels of BRCA1 protein achieving a pathological complete response (pCR), although this was not statistically significant [odds ratio (OR)=1.74; p=0.437]. Twenty-eight percent of patients had PIK3CA mutations, but no statistically significant association with pCR (OR=0.977; p=0.971) was noted. Neither BRCA1 protein levels (OR=1.18; p=0.818) nor PIK3CA mutations (OR=1.03; p=0.971) appeared to be associated with the likelihood of achieving a partial response or better from neoadjuvant chemotherapy. PIK3CA wild-type mutation status showed a trend towards an increased likelihood of not presenting with inflammatory disease (OR=5.34; p=0.101). In this exploratory study, neither BRCA1 protein expression levels nor the presence of PIK3CA mutations were significantly associated with chemotherapy response in locally advanced breast cancer. However, the relatively small sample size limits the overall interpretation.

Repression of cancer cell senescence by PKCι.

Senescence is an irreversible growth arrest phenotype adopted by cells that has a key role in protecting organisms from cancer. There is now considerable interest in therapeutic strategies that reactivate this process to control the growth of cancer cells. Protein kinase-Cι (PKCι) is a member of the atypical PKC family and an important downstream mediator in the phosphoinositide-3-kinase (PI-3-kinase) pathway. PKCι expression was found to be upregulated in a subset of breast cancers and breast cancer cell lines. Activation of the PI-3-kinase pathway by introduction of mutant, oncogenic PIK3CA into breast mammary epithelial cells increased both the expression and activation of PKCι. In breast cancer cells lines overexpressing PKCι, depletion of PKCι increased the number of senescent cells, as assessed by senescence-associated ß-galactosidase, morphology and bromodeoxyuridine incorporation. This phenomenon was not restricted to breast cancer cells, as it was also seen in glioblastoma cells in which PKCι is activated by loss of PTEN. Senescence occurred in the absence of a detectable DNA-damage response, was dependent on p21 and was enhanced by the aurora kinase inhibitor VX-680, suggesting that senescence is triggered by defects in mitosis. Depletion of PKCι had no effect on senescence in normal mammary epithelial cell lines. We conclude that PKCι is overexpressed in a subset of cancers where it functions to suppress premature senescence. This function appears to be restricted to cancer cells and inhibition of PKCι may therefore be an effective way to selectively activate premature senescence in cancer cells.

Regulation of glioblastoma cell invasion by PKC iota and RhoB.

Glioblastoma multiforme is the most aggressive form of primary brain tumor and remains largely incurable, in large part, due to its highly invasive nature. The phosphoinositide (PI) 3-kinase pathway is often constitutively active in these tumors due to activating mutations in the epidermal growth factor receptor, or deletion/loss of function of the tumor suppressor PTEN. Protein kinase C type iota (PKC iota), a member of the atypical protein kinase C family, is activated by the PI 3-kinase pathway and is an important downstream mediator. Here, we have assessed the role of PKC iota in glioblastoma cell invasion. Depletion of PKC iota with RNA interference caused an increase in actin stress fibers and a decrease in cell motility and invasion. Gene expression microarray analysis of U87MG cells showed that PKC iota repressed expression of mRNA for RhoB, which has previously been shown to have a role in actin stress fiber formation. Western blot analysis showed that both PKC iota depletion and pharmacological inhibition of PKC iota caused an increase in the protein levels of RhoB, as did inhibition of PI 3-kinase. Expression of RhoB from a constitutive promoter caused changes in actin stress fibers and cell invasion that were similar to those seen with PKC iota depletion. These data show that PKC iota, activated as a consequence of aberrant upstream PI 3-kinase signaling, mediates glioblastoma cell motility and invasion, and that repression of RhoB is key downstream event in PKC iota signaling leading to enhanced cell motility. In addition, constitutive expression of RhoB repressed PKC iota activity, as assessed by its phosphorylation status on Thr555. PKC iota and RhoB are, therefore, mutually antagonistic, potentially creating a sensitive switch between invasive and non-invasive phenotypes.

Protection of glioblastoma cells from cisplatin cytotoxicity via protein kinase Ciota-mediated attenuation of p38 MAP kinase signaling.

Glioblastoma multiforme is an aggressive form of brain cancer that responds poorly to chemotherapy and is generally incurable. The basis for the poor response of this cancer to chemotherapy is not well understood. The atypical protein kinases C (PKCiota and PKCzeta) have previously been implicated in leukaemia cell chemoresistance. To assess the role of atypical PKC in glioblastoma cell chemoresistance, RNA interference was used to deplete human glioblastoma cells of PKCiota. Transfection of cells with either of two different RNA duplexes specific for PKCiota caused a partial sensitisation to cell death induced by the chemotherapy agent cisplatin. To screen for possible mechanisms for PKCiota-mediated chemoresistance, microarray analysis of gene expression was performed on RNA from glioblastoma cells that were either untreated or depleted of PKCiota. This identified sets of genes that were regulated either positively or negatively by PKCiota. Within the set of genes that were negatively regulated by PKCiota, the function of the gene coding for GMFbeta, an enhancer of p38 mitogen-activated protein kinase (MAP kinase) signaling, was investigated further, as the p38 MAP kinase pathway has been previously identified as a key mediator of cisplatin cytotoxicity. The expression of both GMFbeta mRNA and protein increased upon PKCiota depletion, and this was accompanied by an increase in cisplatin-activated p38 MAP kinase signaling. Transient overexpression of GMFbeta increased cisplatin-activated p38 MAP kinase signaling and also sensitised cells to cisplatin cytotoxicity. The increase in cisplatin cytotoxicity seen with PKCiota depletion was blocked by the p38 MAP kinase inhibitor SKF86002. These data show that PKCiota can confer partial resistance to cisplatin in glioblastoma cells by suppressing GMFbeta-mediated enhancement of p38 MAP kinase signaling.

A phase I study of oral ZD 1839 given daily in patients with solid tumors: IND.122, a study of the Investigational New Drug Program of the National Cancer Institute of Canada Clinical Trials Group.

PURPOSE: To define the maximum tolerated dose (MTD), the dose limiting toxicity (DLT), the biological active (BA) dose and the pharmacokinetics (PK) of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 (Iressa) when administered continuously as a once daily dose in patients with advanced, incurable solid tumours. PATIENTS AND METHODS: Twenty-eight patients were enrolled in cohorts of three from three National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) centers. ZD1839 was given at doses from 150 to 800 mg daily orally and patients underwent a pretreatment and a 28 day post treatment tumor biopsy, while PK sampling was performed on days 8, 15, 22, 29, and a toxicity assessment every 28 days. RESULTS: All twenty-eight patients were evaluable for non-hematological and hematological toxicity. Twenty-seven were evaluable for response. The MTD was not reached but DLT included reversible rash and diarrhea. One patient with urachal cancer had a transient 55% decrease in tumor size and two other patients (breast and non-small cell lung cancer) had minor responses; three additional patients had pharmacodynamic evidence of target effect. PK demonstrated steady state within the first 2 weeks of dosing and dose dependent exposure. CONCLUSION: It appears that ZD 1839 at a dose of 800 m/day was tolerable, although some patients required dose modification for diarrhea. Doses above 250 m/day demonstrate biologic activity and could be consider for future study in a variety of EGFR positive tumor types.