ROLE OF STAT3 IN KERATINOCYTE STEM CELLS DURING SKIN TUMORIGENESIS

STATs play crucial roles in a wide variety of biological functions, including development, proliferation, differentiation, migration and in cancer development. In the present study, we examined the impact of Stat3 deletion or activation on behavior of keratinocytes, including keratinocyte stem cells (KSCs). Deletion of Stat3 specifically in the bulge region of the hair follicle using K15.CrePR1 X Stat3fl/fl mice led to decreased tumor development by altering survival of bulge region KSCs. To further understand the role of KSCs in skin tumorigenesis, K5.Stat3C transgenic (Tg) mice which express a constitutively active/dimerized form of Stat3 called Stat3C via the bovine keratin 5 (K5) promoter were studied. The number of CD34 and α6 integrin positive cells was significantly reduced in Tg mice as compared to non-transgenic (NTg) littermates. There was a concomitant increase in the progenitor populations (Lgr-6, Lrig-1 and Sca-1) in the Tg mice vs. the stem cell population (CD34 and Keratin15). To investigate the mechanism underlying the increase in the progenitor population at the expense of bulge region KSCs we examined if Stat3C expression was involved in inducing migration of the bulge region KSCs. There was altered β-catenin and α6-integrin expression in the hair follicles of Tg mice, which may have contributed to reduced adhesive interactions between the epithelial cells and the basement membrane facilitating migration out of the niche. To further study the effect of Stat3 on differentiation of keratinocytes we analyzed the epidermal keratinocytes in K5.Cre X Stat3fl/fl mice. There was an increase in the expression of epidermal differentiation markers in the Stat3 knockout mice. These data suggest that deletion of Stat3 in the epidermis and hair follicle induced differentiation in these cells. Preliminary studies done with the BK5.Stat3C mouse model suggests that multiple hair follicle stem/progenitor populations may be involved in skin tumor development and progression in this model of skin tumorigenesis. Overall, these data suggest that Stat3 plays an important role in differentiation as well as migration of keratinocytes and that these effects may play a role during epithelial carcinogenesis.

Genome -wide and locus -specific instability

The discovery of expanded simple repeated sequences causing or associated with human disease has lead to a new area of research involved in the elucidation of how the expanded repeat causes disease and how the repeat becomes unstable. ^ To study the genetic basis of the (CTG)n repeat instability in the DMPK gene in myotonic dystrophy (DM1) patients, somatic cell hybrids were constructed between the lymphocytes of DM1 patients and a variety of Chinese hamster ovary (CHO) cell DNA repair gene deficient mutants. By using small pool PCR (SP-PCR), the instability of the (CTG)n can be quantitated for both the frequency and sizes of length change mutations. ^ Additional SP-PCR analysis on 2/11 subclones generated from this original hybrid showed a marked increase in large repeat deletions, ∼50%. A bimodal distribution of repeats was seen around the progenitor allele and at a large deleted product (within the normal range) with no intermediate products present. ^ To determine if the repair capacity of the CHO cell led to a mutator phenotype in the hamster and hybrid clones, SP-PCR was also done on 3 hamster microsatellites in a variety of hamster cell backgrounds. No variant alleles were seen in over 2500 genome equivalents screened. ^ Human-hamster hybrids have long been shown to be chromosomally unstable, yet information about the stability of repeated sequences was not known. To test if repeat instability was associated with either intact or non-intact human chromosomes, more than 300 microsatellite repeats on 13 human chromosomes (intact and non-intact) were analyzed in eight hybrid cells. No variants were seen between the hybrid and patient alleles in the hybrids. ^ To identify whether DM1 patients have a previously undetected level of genome wide instability or if the instability is truly locus specific, SP-PCR was done on 6 human microsatellites within the patient used to make the hybrid cells. No variants were seen in over 1000 genomes screened. ^ These studies show that the somatic cell hybrid approach is a genetically stable system that allows for the determination of factors that could lead to changes in microsatellite instability. It also shows that there is something inherent about the DM1 expanded (CTG)n repeat that it is solely targeted by, as of yet, and unknown mechanism that causes the repeat to be unstable. (Abstract shortened by UMI.)^

Requirement of the Jak2 tyrosine kinase in Bcr -Abl oncogenic transformation

Philadelphia chromosome (Ph)-positive chronic myeloid leukemia is caused by a clonal myeloproliferative expansion of malignant primitive hematopoietic progenitor cells. The Ph results from the reciprocal translocation of the ends of chromosome 9 and 22, which generate Bcr-Abl fusion proteins. The Bcr-Abl proteins possess a constitutively activated Abl tyrosine kinase, which is the driving force responsible for causing leukemia. The activated Bcr-Abl tyrosine kinase stimulates multiple signal transduction pathway affecting growth, differentiation and survival of cells. It is known that the Bcr-Abl tyrosine kinase activates several signaling proteins including Stat5, which is a member of the Jak/Stat pathway that is activated by cytokines that control the growth and differentiation of normal hematopoietic cells. Our laboratory was the first one to report that Jak2 tyrosine kinase is activated in a human Bcr-Abl positive hematopoietic cell line. In this thesis, we further investigated the activation of Jak2 by Bcr-Abl. We found that Jak2 is activated not only in cultured Bcr-abl positive cell lines but also in blood cells from CML blast crisis patients. We also demonstrated that SH2 domain of Bcr-Abl is required for efficient activation Jak2. We further showed that Jak2 binds to the C-terminal domain of Bcr-Abl; tyrosine residue 1007, which is critical for Jak2 activation, is phosphorylated by Bcr-Abl. We searched downstream targets of Jak2 in Bcr-Abl positive cells. We treated Bcr-Abl positive cells with a Jak2 kinase inhibitor AG490 and found that c-Myc protein expression is inhibited by AG490. We further demonstrated that Jak2 inhibitor AG490 not only inhibit C-MYC transcription but also protect c-Myc protein from proteasome-dependent degradation. We also showed that AG490 did not affect Bcr-Abl kinase activity and Stat5 activation and its downstream target Bcl-xL expression. AG490 also induced apoptosis of Bcr-Abl positive cells, similar to Bcr-Abl kinase inhibitor STI571 (also termed Gliveec, a very effective drug for CML), but unlike STI571 the apoptosis effects induced by AG490 can not be rescued by IL-3 containing WEHI conditioned medium. We further established several Bcr-Abl positive clones that express a kinase-inactive Jak2 and found that these clones had reduced tumor formation in nude mice assays. Taken together, these results establish that Jak2 is activated in Bcr-Abl positive CML cells and it is required for c-Myc induction and the oncogenic effects of Bcr-Abl. Furthermore, Jak2 and Stat5 are two independent targets of Bcr-Abl. ^

The role of platelet -derived growth factor (PDGF) signaling in gliomagenesis

Gliomas are primary central nervous system (CNS) neoplasms that are believed to arise from astrocytes, oligodendrocytes or their precursors. Gliomas can be classified into two major histopathological groups: oligodendroglial and astroglial tumors. The most malignant of the astroglial tumors is glioblastoma multiforme (GBM). A great deal of genetic and epigenetic alterations have been implicated in gliomagenesis. In particular, PDGF signaling is frequently over-activated in a large number of human gliomas. In order to gain insights into the biology of gliomas, we manage to model human gliomas in mice using a somatic gene transfer approach—RCAS/TVA system. In our previous study, combined activation of AKT and RAS pathways gave rise to glioblastomas from CNS progenitors. In the present study, we demonstrate that in vivo autocrine PDGF stimulation induces oligodendrogliomas and mixed oligoastrocytomas from CNS progenitors and differentiated astrocytes respectively. In culture autocrine PDGF stimulation dedifferentiates astrocytes into progenitor-like cells and blockade of PDGF signaling reverses these phenotypic changes. Experimental disruption of cell cycle arrest pathway, such as Ink4a-Arf loss, is not required for the initiation of PDGF-induced gliomagenesis; instead, this mutation contributes to the tumor progression by enhancing tumor malignancy and shortening tumor latency. P53 deficiency does not promote the PDGF-induced gliomagenesis. In addition, 1p and 19q, often deleted in human oligodendrogliomas, remain intact in these PDGF-induced gliomas. Therefore, our studies suggest that autocrine PDGF stimulation alone may be sufficient to induce gliomagenesis. In contrast to transient stimulation in vitro, constitutive PDGF stimulation activates neither AKT nor RAS/MAPK pathways during gliomagenesis. This results in the formation of oligodendrogliomas, instead of glioblastomas. Sustained activation of the AKT pathway converts PDGF-induced oligodendrogliomas into astrocytomas. Our studies suggest that constitutive PDGF stimulation is not equivalent to transient PDGF stimulation, and that a transition between oligodendroglial and astroglial tumors in humans may be possible, depending on additional alterations. In summary, PDGF signaling plays a pivotal role in gliomagenesis in the mouse, and its hyperactivity is capable of contributing to both oligodendroglial and astroglial tumorigenesis. ^