Overexpression of the type II transmembrane serine protease matriptase is a highly consistent feature of human epithelial tumors. Here we show that matriptase possesses a strong oncogenic potential when unopposed by its endogenous inhibitor, HAI-1. Modest orthotopic overexpression of matriptase in the skin of transgenic mice caused spontaneous squamous cell carcinoma and dramatically potentiated carcinogen-induced tumor formation. Matriptase-induced malignant conversion was preceded by progressive interfollicular hyperplasia, dysplasia, follicular transdifferentiation, fibrosis, and dermal inflammation. Furthermore, matriptase induced activation of the pro-tumorigenic PI3K-Akt signaling pathway. This activation was frequently accompanied by H-ras or K-ras mutations in carcinogen-induced tumors, whereas matriptase-induced spontaneous carcinoma formation occurred independently of ras activation. Increasing epidermal HAI-1 expression completely negated the oncogenic effects of matriptase. The data implicate dysregulated matriptase expression in malignant epithelial transformation.

The role of the BCR–ABL oncogene in the progression of chronic myeloid leukemia (CML) to blast crisis (BC) is unknown. The appearance of chromosomal aberrations in patients with CML BC has led to many attempts to elucidate a mechanism whereby BCR–ABL affects DNA damage and repair. BCR–ABL-expressing cells have been found to accumulate genetic abnormalities, but the mechanism leading to this genomic instability is controversial. In this study, we review the effects of BCR–ABL on DNA repair mechanisms, centrosomes, checkpoint activation and apoptosis. BCR–ABL has diverse effects on these mechanisms, but which of these effects are necessary for the progression of CML to BC is still unresolved.

Earlier reports have suggested that the BCR/ABL oncogene, associated with chronic myeloid leukemia, induces a mutator phenotype; however, it is unclear whether this leads to long-term changes in chromosomes and whether the phenotype is found in primary chronic myelogeneous leukemia (CML) cells. We have addressed both these issues. BCR/ABL-expressing cell lines show an increase in DNA breaks after treatment with etoposide as compared to control cells. However, although BCR/ABL-expressing cell lines have an equivalent cell survival, they have an increase in chromosomal translocations after DNA repair as compared to control cells. This demonstrates that BCR/ABL expression decreases the fidelity of DNA repair. To see whether this is true in primary CML samples, normal CD34 + progenitor cells and CML progenitor cells were treated with etoposide. CML progenitor cells have equivalent survival but have an increase in DNA double-strand breaks (DSBs). Spectral karyotyping demonstrates new chromosomal translocations in CML cells, but not normal progenitor cells, consistent with error-prone DNA repair. Taken together, these data demonstrate that BCR/ABL enhances the accumulation of DSBs and alters the apoptotic threshold in CML leading to error-prone DNA repair.

Recent results have demonstrated that multiple signal transduction pathways are activated in acute myeloid leukemia (AML) cells, however, the tyrosine kinase(s) that phosphorylates these signaling proteins is not identified. We have analyzed AML cells using a phosphoproteomics screen and demonstrate that the Src family kinases, Lyn, Lck and Fgr, are phosphorylated on their activation sites in AML samples. Expression and activation of Lyn has been previously confirmed. Evaluation of Lck demonstrated that Lck is expressed to a variable degree but consistently in AML samples (n=20). Lck kinase assays show activation of Lck in 17/20 samples tested at levels above the level of activation detectable in normal CD34+ progenitor cells. Lyn and Lck both contribute to AML cell growth as siRNA depletion of either kinase leads to decreased leukemia colony forming activity. Interestingly, both Lyn and Lck contribute to phosphorylation of STAT5 as STAT5 phosphorylation is decreased but not abrogated by siRNA modification of either kinase alone. Consistent with the necessity for this signaling pathway for optimal AML cell growth, siRNA knockdown of STAT5 leads to decreased expression of both STAT5A and STAT5B, decreased expression of the STAT5 target protein, Bclxl and decreased AML colony forming ability. Based on this data, we have studied the FDA approved compound, Dasatinib, and demonstrate that Dasatinib decreases AML colony formation in 4 out of 5 samples tested. Overall, these results demonstrate that SFK’s act redundantly to regulate STAT5 phosphorylation and AML cell growth in primary cells and that phosphoprotein analysis is a robust approach to identify new targets for therapy of malignancy. Src family kinase inhibitors may be valuable in the therapy of AML.

Chronic myeloid leukemia (CML) is a two-stage disease associated with the t(9;22) translocation. This translocation fuses the BCR gene with the ABL tyrosine kinase, forming the BCR/ABL oncogene. BCR/ABL is a constitutively activated tyrosine kinase, which causes the first stage of CML, chronic phase. However, it is still unknown why patients progress to the second phase, blast crisis, a phase marked by increased chromosomal abnormalities. Previous data from our lab suggests that cells expressing BCR/ABL have an increase in chromosomal translocations after DNA repair compared to control cells, as assessed by spectral karyotyping. We hypothesized that BCR/ABL alters the apoptotic threshold in response to DNA damage, and we sought to determine if cells lacking BCR/ABL that were unable to undergo apoptosis would show similar responses in DNA damage and repair assays to BCR/ABL expressing cells. In order to study this question we have used an interleukin 3 (IL3) dependent cell line that lacks expression of the pro-apoptotic proteins, Bax and Bak. These cells grow in the presence of IL3 but do not undergo apoptosis after cytokine withdrawal or genotoxic stress. We have generated a subclone of these cells that expresses BCR/ABL. This cell line, designated DBA, grows in the absence of IL3. As expected, growth of the cells in the absence of IL3 is suppressed by the ABL kinase inhibitor, dasatinib. BCR/ABL expressing and control cells proliferate at similar rates following DNA damage. After damage, both control cells and DBA cells have a delay in the G2/M phase of the cell cycle, which is modestly prolonged in BCR/ABL expressing cells. Both control and BCR/ABL expressing cells have a similar amount of DNA damage after irradiation as assessed by pulsed field gel electrophoresis. Additionally, the rate of repair of DNA double strand breaks is not significantly different in the two cell types. Spectral karyotype analysis is ongoing to determine whether error prone DNA repair in BCR/ABL cells is secondary to the effects of BCR/ABL on inhibition of apoptosis or reflects other proposed mechanisms of action of the oncogene.