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Ectopic expression of mutant forms of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) lacking lipid (G129E) or lipid and protein (C124S) phosphatase activity decreased sensitivity of MCF-7 breast cancer cells, which have wild-type PTEN, to doxorubicin and increased sensitivity to the mammalian target of rapamycin (mTOR) inhibitor rapamycin. Cells transfected with a mutant PTEN gene lacking both lipid and protein phosphatase activities were more resistant to doxorubicin than cells transfected with the PTEN mutant lacking lipid phosphatase activity indicating that the protein phosphatase activity of PTEN was also important in controlling the sensitivity to doxorubicin, while no difference was observed between the lipid (G129E) and lipid and protein (C124S) phosphatase PTEN mutants in terms of sensitivity to rapamycin. A synergistic inhibitory interaction was observed when doxorubicin was combined with rapamycin in the phosphatase-deficient PTENtransfected cells. Interference with the lipid phosphatase activity of PTEN was sufficient to activate Akt/mTOR/ p70S6K signaling. These studies indicate that disruption of the normal activity of the PTEN phosphatase can have dramatic effects on the therapeutic sensitivity of breast cancer cells. Mutations in the key residues which control PTEN lipid and protein phosphatase may act as dominant-negative mutants to suppress endogenous PTEN and alter the sensitivity of breast cancer patients to chemo-and targeted therapies.

Targeting MEK has been shown to be effective in suppressing many important pathways involved in cell growth and the prevention of apoptosis. MEK inhibitors have many potential therapeutic uses in the suppression of cancer, proliferative diseases and aging.

Various Raf inhibitors have been developed and are being clinically used to treat patients with melanoma, thyroid, hepatocellular and renal cell cancers. Some 'Raf-kinase inhibitors' affect other kinases which are also expressed on malignant cells; yet, these inhibitors have proven useful in the therapy of certain cancer patients. Other more recently developed Raf specific inhibitors have shown success in the treatment of tumors bearing Raf mutations. The development of Raf inhibitors has significantly advanced cancer therapy in the past decade.

ABSTRACTv-ErbB is an oncogene related to the Epidermal Growth Factor Receptor (EGFR). EGFR overexpression has been observed in many pathological situations. There is a truncated form of EGFR, referred to as EGFvIII, which resembles v-ErbB in biological properties and is often expressed in certain human tumors. Aberrant EGFR expression in human cancers is often constitutive and may occur in the presence of mutated oncogenes or tumor suppressor genes. To circumvent these problems, we subcloned v-ErbB into a vector which contains the estrogen receptor hormone binding domain (ER) which renders the v-ErbB:ER protein dependent upon β-estradiol for activity. v-ErbB:ER conditionally abrogated the cytokine dependence of hematopoietic cells more efficiently than activated v-Ha-Ras, v-Src, Raf or Akt. Abrogation of cytokine-dependence by v-ErbB:ER was not due to the synthesis of autocrine growth factors. Treatment of v-ErbB:ER cells with the EGFR inhibitor AG1478 efficiently induced apoptosis. Induction of apoptosis and prevention of cell cycle progression by the EGFR inhibitor were only observed when the cells were grown in response to v-ErbB:ER activation demonstrating specificity. In contrast, the other inhibitors suppressed cell cycle progression when the cells were grown in response to v-ErbB:ER or the cytokine interleukin-3. When MEK and either EGFR or PI3K/mTOR inhibitors were added, an enhanced apoptotic response was observed. Thus this conditional ErbB construct is useful to elucidate EGFR signaling and anti-apoptotic pathways in the absence of autocrine cytokine expression.

Epidermal growth factor (EGF) is commonly thought to affect the proliferation of many cells, especially epithelial cells. Aberrant expression of the receptor for EGF, (EGFR) or members of the EGFR family is often implicated in the etiology of many cancers. Ligation of the EGFR results in the activation of many downstream signaling pathways which have profound effects on cell cycle progression and the prevention of apoptosis. In general, the EGFR is thought to be either not expressed or expressed at low levels in hematopoietic cells. We determined that the EGFR was expressed at a low level in the murine cytokine-dependent hematopoietic cell line FDC-P1 but not in an additional murine IL-3 dependent cell line FL5. EGF induced a mild effect on DNA synthesis and ERK activation in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Addition of suboptimal concentrations of IL-3 synergized with EGF in stimulating DNA synthesis in EGFR-positive FDC-P1 cells. Likewise, the EGFR inhibitor AG1478 induced apoptosis in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Both cell lines can be directly transformed to cytokine independence by activated EGFR (v-ERBB) expression in the absence of autocrine growth factors indicating that they are poised to fully utilize EGFR mediated signal transduction pathways as a means for proliferation. These results document the functional importance of endogenous EGFR signaling pathway in some hematopoietic cells.

Mutations in the Ras pathway have been detected in approximately 50% of hematopoietic malignancies. These mutations usually occur at KRAS, NRAS or upstream FLT3. Recent results from an Asian based leukemia study indicated that the BRAF gene is mutated in 4% of AMLs and 3% of NHL. We sought to determine whether the BRAF, KRAS, PI3K genes were mutated in various hematopoietic malignancies of European ancestry as differences in mutation frequencies for other genes has been reported among various ethnic groups. As controls for BRAF mutations, the structure of the BRAF gene was examined in melanoma biopsies from European patients with various stages of melanoma. 61% of the melanoma patients had the BRAF V600E mutation. In contrast, essentially no mutations were detected in the European based NHL and AML patients. The presence of CMV infection in NHL did not change the genetic status of BRAF. To determine the effects of the BRAF V600E mutation on the cytokine-dependency of hematopoietic cells, cDNAs encoding BRAF wild type (WT) and BRAF V600E mutation were ligated to the hormone binding domain of a mutated estrogen receptor (ER*) which responds to 4 hydroxyl tamoxifen (4HT). These modified cDNAs were inserted into retroviral vectors. BRAF V600E induced the morphological transformation of NIH-3T3 cells in a 4HT-dependent fashion. Prolonged expression of BRAF V600E induced anoikis, results consistent with our previous studies indicating that BRAF overexpression can induce cell cycle arrest and apoptosis. Three cytokine dependent hematopoietic cell lines (human TF-1, murine FL5.12 and FDC-P1) were infected with WT and V600E BRAF encoding retroviruses. Expression of WT or V600E BRAF did not efficiently abrogate cytokine dependence. To determine if expression of the PI3K/Akt pathway could synergize with BRAF to induce cytokine independence, WT and V600E BRAF transduced cells were infected with WT and activated PI3K (p110 catalytic subunit), active and inactive Akt and an empty retroviral vector as a control. Conditional (Akt:ER*Myr+) and constitutive (Akt−Myr+) and PI3K but not inactive (Akt:ER*Myr−) synergized at least 250-fold with activated V600E but not WT BRAF to abrogate cytokine-dependence. Conditional activation of BRAF V600E induced downstream MEK and ERK, which was associated with cell cycle progression and prevention of both caspase 3 activation and apoptosis. Prevention of apoptosis in the BRAF V600E and Akt transformed hematopoietic cells was highly sensitive to 3 different MEK inhibitors and apoptosis was synergistically enhanced when the cells were treated with MEK and the mTOR inhibitor rapamycin. Our results indicate that the BRAF gene is infrequently mutated in hematopoietic malignancies and that the BRAF V600E mutant does not efficiently abrogate the cytokine dependence of 3 hematopoietic cell lines. However activated V600E but not WT BRAF synergized with the activated PI3K/Akt pathway to induce cytokine independence which was MEK1 dependent. Our data suggest that the BRAF mutation may not be detected frequently in hematopoietic malignancies because it is relatively inefficient by itself in conferring a growth advantage to hematopoietic cells (e.g., cytokine-independence) which is often associated with the induction of leukemia.