Appearance of tuft cells during prostate cancer progression.

Tuft cells are chemosensory epithelial cells that increase in number following infection or injury to robustly activate the innate immune response to alleviate or promote disease. Recent studies of castration resistant prostate cancer and its subtype, neuroendocrine prostate cancer, revealed Pou2f3+ populations in mouse models. The transcription factor Pou2f3 is a master regulator of the tuft cell lineage. We show that tuft cells are upregulated early during prostate cancer development, and their numbers increase with progression. Cancer-associated tuft cells in the mouse prostate express DCLK1, COX1, COX2, while human tuft cells express COX1. Mouse and human tuft cells exhibit strong activation of signaling pathways including EGFR and SRC-family kinases. While DCLK1 is a mouse tuft cell marker, it is not present in human prostate tuft cells. Tuft cells that appear in mouse models of prostate cancer display genotype-specific tuft cell gene expression signatures. Using bioinformatic analysis tools and publicly available datasets, we characterized prostate tuft cells in aggressive disease and highlighted differences between tuft cell populations. Our findings indicate that tuft cells contribute to the prostate cancer microenvironment and may promote development of more advanced disease. Further research is needed to understand contributions of tuft cells to prostate cancer progression.

Protein tyrosine kinase 6 regulates activation of SRC kinase.

Expression of Protein tyrosine kinase 6 (PTK6) is upregulated in several human solid tumors, and it has oncogenic roles in prostate and breast cancer. PTK6 and SRC kinase are distantly related, share many substrates, and often regulate the same signaling pathways, but whether they interact to regulate signaling is not well understood. We characterized crosstalk between PTK6 and SRC and show that PTK6 can directly phosphorylate SRC to promote its activation. Stable knockdown of PTK6 in multiple cancer cell lines leads to decreased activating phosphorylation of SRC. We show that coexpression of kinase-dead SRC and active PTK6 in mouse embryonic fibroblasts lacking Src, Yes, and Fyn results in activating phosphorylation of SRC. However, there is no reciprocal effect, because active SRC does not promote activating phosphorylation of PTK6. Overexpression of active PTK6 maintained activation of epidermal growth factor receptor (EGFR), AKT, and FAK, but not SHP2 and ERK1/2 in cells with knockdown of SRC. Both PTK6 and SRC are regulated by EGFR, and its inhibition with erlotinib downregulated PTK6 and to a lesser degree SRC activation in LNCaP cells that overexpress active PTK6. Erlotinib treatment also led to AKT inhibition, but overexpression of active PTK6 prevented this. Our data demonstrate overlapping and unique functions for PTK6 and SRC. Finally, we show that PTK6 and SRC are coexpressed in subsets of human prostate and breast cancer cells, and active PTK6 and active SRC colocalize in prostate cancer, supporting a role for PTK6 in promoting SRC activity in cancer.

Targeting protein tyrosine kinase 6 in cancer.

Protein tyrosine kinase 6 (PTK6) is the most well studied member of the PTK6 family of intracellular tyrosine kinases. While it is expressed at highest levels in differentiated cells in the regenerating epithelial linings of the gastrointestinal tract and skin, induction and activation of PTK6 is detected in several cancers, including breast and prostate cancer where high PTK6 expression correlates with worse outcome. PTK6 expression is regulated by hypoxia and cell stress, and its kinase activity is induced by several growth factor receptors implicated in cancer including members of the ERBB family, IGFR1 and MET. Activation of PTK6 at the plasma membrane has been associated with the epithelial mesenchymal transition and tumor metastasis. Several lines of evidence indicate that PTK6 has context dependent functions that depend on cell type, intracellular localization and kinase activation. Systemic disruption of PTK6 has been shown to reduce tumorigenesis in mouse models of breast and prostate cancer, and more recently small molecule inhibitors of PTK6 have exhibited efficacy in inhibiting tumor growth in animal models. Here we review data that suggest targeting PTK6 may have beneficial therapeutic outcomes in some cancers.

Protein tyrosine kinase 6 signaling in prostate cancer.

More than 25 years have passed since the discovery of protein tyrosine kinase 6 (PTK6), a non-receptor tyrosine kinase distantly related to SRC family kinases. Since then, a variety of data suggest that PTK6 promotes oncogenic signaling and tumorigenesis, generally dependent on its kinase activity. Increased PTK6 expression, activation at the plasma membrane and altered intracellular localization have been discovered in prostate cancers. While PTK6 is localized to nuclei of epithelial cells in normal prostate, it is relocalized and activated at the plasma membrane in prostate tumors. Active PTK6 interacts with and directly phosphorylates AKT, FAK and BCAR1 to promote oncogenic signaling. Furthermore, PTK6 can enhance the epithelial mesenchymal transition by inhibiting E-cadherin expression and inducing expression of the mesenchymal markers vimentin, SLUG and ZEB1. Several lines of evidence suggest that PTK6 plays a role in Pten null prostate tumors. PTEN targets activating phosphorylation of PTK6 and loss of PTEN subsequently leads to PTK6 activation. Different studies provide compelling evidence as to why PTK6 is a potential therapeutic target in prostate cancer. Here, we briefly review the advances and significance of PTK6 in prostate cancer.

Vemurafenib Inhibits Active PTK6 in PTEN-null Prostate Tumor Cells.

Protein tyrosine kinase 6 (PTK6, also called BRK) is overexpressed and activated in human prostate cancer. Loss of the tumor suppressor PTEN, a frequent event in prostate cancer, leads to PTK6 activation at the plasma membrane and its oncogenic signaling. The small molecule inhibitor vemurafenib, also known as PLX4032, and its tool analog PLX4720 were designed to inhibit constitutively active BRAF V600E, yet they also have potent effects against PTK6. Vemurafenib is used in the treatment of metastatic melanoma, but its efficacy in prostate cancer has not been assessed. When activated at the plasma membrane, PTK6 promotes signaling through FAK, EGFR, and ERK1/2, and we show this can be blocked by vemurafenib. In addition, PTK6-mediated cell growth, migration, and invasion are inhibited upon vemurafenib administration. Using a flank xenograft model, vemurafenib treatment reduced tumor burden. Using saturation transfer difference NMR and molecular docking, we demonstrate that vemurafenib binds in the active site of PTK6, inhibiting its activation. These structural studies provide insight into the PTK6-vemurafenib complex, which can be utilized for further refinement chemistry, whereas functional studies demonstrate that active PTK6 is a viable drug target in prostate cancer.

PTEN is a protein phosphatase that targets active PTK6 and inhibits PTK6 oncogenic signaling in prostate cancer.

PTEN activity is often lost in prostate cancer. We show that the tyrosine kinase PTK6 (BRK) is a PTEN substrate. Phosphorylation of PTK6 tyrosine 342 (PY342) promotes activation, while phosphorylation of tyrosine 447 (PY447) regulates auto-inhibition. Introduction of PTEN into a PTEN null prostate cancer cell line leads to dephosphorylation of PY342 but not PY447 and PTK6 inhibition. Conversely, PTEN knockdown promotes PTK6 activation in PTEN positive cells. Using a variety of PTEN mutant constructs, we show that protein phosphatase activity of PTEN targets PTK6, with efficiency similar to PTP1B, a phosphatase that directly dephosphorylates PTK6 Y342. Conditional disruption of Pten in the mouse prostate leads to tumorigenesis and increased phosphorylation of PTK6 Y342, and disruption of Ptk6 impairs tumorigenesis. In human prostate tumor tissue microarrays, loss of PTEN correlates with increased PTK6 PY342 and poor outcome. These data suggest PTK6 activation promotes invasive prostate cancer induced by PTEN loss.

Kinase-Dependent and -Independent Roles for PTK6 in Colon Cancer.

UNLABELLED: Disruption of the gene encoding Protein Tyrosine Kinase 6 (Ptk6) delayed differentiation and increased growth in the mouse intestine. However, Ptk6-null mice were also resistant to azoxymethane-induced colon tumorigenesis. To further explore functions of PTK6 in colon cancer, expression of epithelial and mesenchymal markers, as well as proliferation, migration, and xenograft tumor growth, was examined in human colon tumor cell lines with knockdown or overexpression of PTK6. PTK6 protein, transcript, and activation were also examined in a human colon tumor tissue array, using immunohistochemistry and qRT-PCR. Knockdown of PTK6 led to the epithelial-mesenchymal transition (EMT) in SW480 and HCT116 cells, whereas overexpression of PTK6 in SW620 cells restored an epithelial phenotype in a kinase-independent manner. PTK6 knockdown also increased xenograft tumor growth of SW480 cells, suggesting tumor suppressor functions. In clinical specimens, PTK6 expression was highest in normal differentiated epithelial cells and reduced in tumors. In contrast, overexpression of constitutively active PTK6 promoted STAT3 and ERK5 activation in colon cancer cells, and endogenous PTK6 promoted cell survival and oncogenic signaling in response to DNA-damaging treatments. These data indicate that PTK6 has complex, context-specific functions in colon cancer; PTK6 promotes the epithelial phenotype to antagonize the EMT in a kinase-independent manner, whereas activation of PTK6 promotes oncogenic signaling. IMPLICATIONS: Understanding context-specific functions of PTK6 is important, because although it promotes cell survival and oncogenic signaling after DNA damage, expression of PTK6 in established tumors may maintain the epithelial phenotype, preventing tumor progression. Mol Cancer Res; 14(6); 563-73. ©2016 AACR.

Caveolin-1 regulates cancer cell metabolism via scavenging Nrf2 and suppressing MnSOD-driven glycolysis.

Aerobic glycolysis is an indispensable component of aggressive cancer cell metabolism. It also distinguishes cancer cells from most healthy cell types in the body. Particularly for this reason, targeting the metabolism to improve treatment outcomes has long been perceived as a potentially valuable strategy. In practice, however, our limited knowledge of why and how metabolic reprogramming occurs has prevented progress towards therapeutic interventions that exploit the metabolic peculiarities of tumors. We recently described that in breast cancer, MnSOD upregulation is both necessary and sufficient to activate glycolysis. Here, we focused on determining the molecular mechanisms of MnSOD upregulation. We found that Caveolin-1 (Cav-1) is a central component of this mechanism due to its suppressive effects of NF-E2-related factor 2 (Nrf2), a transcription factor upstream of MnSOD. In transformed MCF10A(Er/Src) cells, Cav-1 loss preceded the activation of Nrf2 and its induction of MnSOD expression. Consistently, with previous observations, MnSOD expression secondary to Nrf2 activation led to an increase in the glycolytic rate dependent on mtH2O2 production and the activation of AMPK. Moreover, rescue of Cav-1 expression in a breast cancer cell line (MCF7) suppressed Nrf2 and reduced MnSOD expression. Experimental data were reinforced by epidemiologic nested case-control studies showing that Cav-1 and MnSOD are inversely expressed in cases of invasive ductal carcinoma, with low Cav-1 and high MnSOD expression being associated with lower 5-year survival rates and molecular subtypes with poorest prognosis.

Protein Tyrosine Kinase 6 Regulates UVB-Induced Signaling and Tumorigenesis in Mouse Skin.

Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed in the epithelial linings of the gastrointestinal tract and the skin, where it is expressed in nondividing differentiated cells. We found that PTK6 expression increases in the epidermis following UVB treatment. To evaluate the roles of PTK6 in the skin following UVB-induced damage, we exposed back skin of Ptk6 +/+ and Ptk6 -/- SENCAR mice to incremental doses of UVB for 30 weeks. Wild-type mice were more sensitive to UVB and exhibited increased inflammation and greater activation of signal transducer and activator of transcription-3 (STAT3) than Ptk6-/- mice. Disruption of Ptk6 did not have an impact on proliferation, although PTK6 was expressed and activated in basal epithelial cells in wild-type mice following UVB treatment. However, wild-type mice exhibited shortened tumor latency and increased tumor load compared with Ptk6-/- mice, and STAT3 activation was increased in these tumors. PTK6 activation was detected in UVB-induced tumors, and this correlated with increased activating phosphorylation of focal adhesion kinase (FAK) and breast cancer anti-estrogen resistance 1 (BCAR1). Activation of PTK6 was also detected in human squamous cell carcinomas of the skin. Although PTK6 has roles in normal differentiation, it also contributes to UVB-induced injury and tumorigenesis in vivo.

Brk/Protein tyrosine kinase 6 phosphorylates p27KIP1, regulating the activity of cyclin D-cyclin-dependent kinase 4.

Cyclin D and cyclin-dependent kinase 4 (cdk4) are overexpressed in a variety of tumors, but their levels are not accurate indicators of oncogenic activity because an accessory factor such as p27(Kip1) is required to assemble this unstable dimer. Additionally, tyrosine (Y) phosphorylation of p27 (pY88) is required to activate cdk4, acting as an "on/off switch." We identified two SH3 recruitment domains within p27 that modulate pY88, thereby modulating cdk4 activity. Via an SH3-PXXP interaction screen, we identified Brk (breast tumor-related kinase) as a high-affinity p27 kinase. Modulation of Brk in breast cancer cells modulates pY88 and increases resistance to the cdk4 inhibitor PD 0332991. An alternatively spliced form of Brk (Alt Brk) which contains its SH3 domain blocks pY88 and acts as an endogenous cdk4 inhibitor, identifying a potentially targetable regulatory region within p27. Brk is overexpressed in 60% of breast carcinomas, suggesting that this facilitates cell cycle progression by modulating cdk4 through p27 Y phosphorylation. p27 has been considered a tumor suppressor, but our data strengthen the idea that it should also be considered an oncoprotein, responsible for cyclin D-cdk4 activity.

PTK6/BRK is expressed in the normal mammary gland and activated at the plasma membrane in breast tumors.

Protein Tyrosine kinase 6 (PTK6/BRK) is overexpressed in the majority of human breast tumors and breast tumor cell lines. It is also expressed in normal epithelial linings of the gastrointestinal tract, skin, and prostate. To date, expression of PTK6 has not been extensively examined in the normal human mammary gland. We detected PTK6 mRNA and protein expression in the immortalized normal MCF-10A human mammary gland epithelial cell line, and examined PTK6 expression and activation in a normal human breast tissue microarray, as well as in human breast tumors. Phosphorylation of tyrosine residue 342 in the PTK6 activation loop corresponds with its activation. Similar to findings in the prostate, we detect nuclear and cytoplasmic PTK6 in normal mammary gland epithelial cells, but no phosphorylation of tyrosine residue 342. However, in human breast tumors, striking PTK6 expression and phosphorylation of tyrosine 342 is observed at the plasma membrane. PTK6 is expressed in the normal human mammary gland, but does not appear to be active and may have kinase-independent functions that are distinct from its cancer promoting activities at the membrane. Understanding consequences of PTK6 activation at the plasma membrane may have implications for developing novel targeted therapies against this kinase.

PTK6 activation at the membrane regulates epithelial-mesenchymal transition in prostate cancer.

The intracellular tyrosine kinase protein tyrosine kinase 6 (PTK6) lacks a membrane-targeting SH4 domain and localizes to the nuclei of normal prostate epithelial cells. However, PTK6 translocates from the nucleus to the cytoplasm in human prostate tumor cells. Here, we show that while PTK6 is located primarily within the cytoplasm, the pool of active PTK6 in prostate cancer cells localizes to membranes. Ectopic expression of membrane-targeted active PTK6 promoted epithelial-mesenchymal transition in part by enhancing activation of AKT, thereby stimulating cancer cell migration and metastases in xenograft models of prostate cancer. Conversely, siRNA-mediated silencing of endogenous PTK6 promoted an epithelial phenotype and impaired tumor xenograft growth. In mice, PTEN deficiency caused endogenous active PTK6 to localize at membranes in association with decreased E-cadherin expression. Active PTK6 was detected at membranes in some high-grade human prostate tumors, and PTK6 and E-cadherin expression levels were inversely correlated in human prostate cancers. In addition, high levels of PTK6 expression predicted poor prognosis in patients with prostate cancer. Our findings reveal novel functions for PTK6 in the pathophysiology of prostate cancer, and they define this kinase as a candidate therapeutic target. Cancer Res; 73(17); 5426-37. ©2013 AACR.

Targeting FoxM1 effectively retards p53-null lymphoma and sarcoma.

The forkhead box transcription factor FOXM1 is considered to be a promising target for cancer therapy. However, the significance of FOXM1 in tumors harboring mutation in p53, which is very common, is unclear. In this study, we investigated the efficacy of FoxM1 targeting in spontaneous p53-null tumors using genetic ablation as well as using a peptide inhibitor of FOXM1. We show that conditional deletion of FoxM1 inhibits growth of the p53-null thymic lymphoma and sarcoma cells. In addition, deletion of FoxM1 induces apoptotic cell death of the p53-null tumors, accompanied by reduced expression of the FOXM1 target genes survivin and Bmi1. An ARF-derived peptide that inhibits the activity of FOXM1, by targeting it to the nucleolus, also induces apoptosis in the p53-null sarcoma and lymphoma, leading to a strong inhibition of their metastatic colonization. Together, our observations suggest that FOXM1 is critical for survival and growth of the p53-null lymphoma and sarcoma and provide proof-of-principle that FOXM1 is an effective therapeutic target for sarcoma and lymphoma carrying loss of function mutation in p53.

Context-specific protein tyrosine kinase 6 (PTK6) signalling in prostate cancer.

BACKGROUND: Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is distantly related to SRC family kinases. PTK6 is nuclear in normal prostate epithelia, but nuclear localization is lost in prostate tumours. Increased expression of PTK6 is detected in human prostate cancer, especially at metastatic stages, and in other types of cancers, including breast, colon, head and neck cancers, and serous carcinoma of the ovary. MATERIALS AND METHODS: Potential novel substrates of PTK6 identified by mass spectrometry were validated in vitro. The significance of PTK6-induced phosphorylation of these substrates was addressed using human prostate cell lines by knockdown of endogenous PTK6 or overexpression of targeted PTK6 to different intracellular compartments. RESULTS: We identified AKT, p130CAS and focal adhesion kinase (FAK) as novel PTK6 substrates and demonstrated their roles in promoting cell proliferation, migration and resistance to anoikis. In prostate cancer cells, active PTK6 is primarily associated with membrane compartments, although the majority of total PTK6 is localized within the cytoplasm. Ectopic expression of membrane-targeted PTK6 transforms immortalized fibroblasts. Knockdown of endogenous cytoplasmic PTK6 in PC3 prostate cancer cells impairs proliferation, migration and anoikis resistance. However, re-introduction of PTK6 into the nucleus significantly decreases cell proliferation, suggesting context-specific functions for nuclear PTK6. CONCLUSIONS: In human prostate cancer, elevated PTK6 expression, translocation of PTK6 from the nucleus to the cytoplasm and its activation at the plasma membrane contribute to increased phosphorylation and activation of its substrates such as AKT, p130CAS and FAK, thereby promoting prostate cancer progression.

Targeting protein tyrosine kinase 6 enhances apoptosis of colon cancer cells following DNA damage.

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that has distinct functions in normal epithelia and cancer. It is expressed primarily in nondividing epithelial cells in the normal intestine, where it promotes differentiation. However, after DNA damage, PTK6 is induced in proliferating progenitor cells, where it contributes to apoptosis. We examined links between PTK6 and the tumor suppressor p53 in the isogenic p53(+/+) and p53(-/-) HCT116 colon tumor cell lines. We found that p53 promotes expression of PTK6 in HCT116 cells, and short hairpin RNA-mediated knockdown of PTK6 leads to reduced induction of the cyclin-dependent kinase inhibitor p21. Knockdown of PTK6 enhances apoptosis in HCT116 cells with wild-type p53, following treatment of cells with γ-radiation, doxorubicin, or 5-fluorouracil. No differences in the activation of AKT, ERK1/2, or ERK5, known PTK6-regulated prosurvival signaling proteins, were detected. However, activity of STAT3, a PTK6 substrate, was impaired in cells with knockdown of PTK6 following DNA damage. In contrast to its role in the normal epithelium following DNA damage, PTK6 promotes survival of cancer cells with wild-type p53 by promoting p21 expression and STAT3 activation. Targeting PTK6 in combination with use of chemotherapeutic drugs or radiation may enhance death of colon tumor cells with wild-type p53.

Protein-tyrosine kinase 6 promotes peripheral adhesion complex formation and cell migration by phosphorylating p130 CRK-associated substrate.

Protein-tyrosine kinase 6 (PTK6) is a non-myristoylated intracellular tyrosine kinase evolutionarily related to Src kinases. Aberrant PTK6 expression and intracellular localization have been detected in human prostate tumors. In the PC3 prostate cancer cell line, the pool of endogenous activated PTK6, which is phosphorylated on tyrosine residue 342, is localized at the membrane. Expression of ectopic membrane-targeted PTK6 led to dramatic morphology changes and formation of peripheral adhesion complexes in PC3 cells. Peripheral adhesion complex formation was dependent upon PTK6 kinase activity. We demonstrated that p130 CRK-associated substrate (p130CAS) is a novel direct substrate of PTK6, and it works as a crucial adapter protein in inducing peripheral adhesion complexes. Activation of ERK5 downstream of p130CAS was indispensable for this process. Knockdown of endogenous PTK6 led to reduced cell migration and p130CAS phosphorylation, whereas knockdown of p130CAS attenuated oncogenic signaling induced by membrane-targeted PTK6, including ERK5 and AKT activation. Expression of membrane-targeted PTK6 promoted cell migration, which could be impaired by knockdown of p130CAS or ERK5. Our study reveals a novel function for PTK6 at the plasma membrane and suggests that the PTK6-p130CAS-ERK5 signaling cascade plays an important role in cancer cell migration and invasion.

Disruption of the mouse protein tyrosine kinase 6 gene prevents STAT3 activation and confers resistance to azoxymethane.

BACKGROUND & AIMS: Protein tyrosine kinase 6 (PTK6) is expressed throughout the gastrointestinal tract and is a negative regulator of proliferation that promotes differentiation and DNA-damage-induced apoptosis in the small intestine. PTK6 is not expressed in normal mammary gland, but is induced in most human breast tumors. Signal transducer and activator of transcription 3 (STAT3) mediates pathogenesis of colon cancer and is a substrate of PTK6. We investigated the role of PTK6 in colon tumorigenesis. METHODS: Ptk6+/+ and Ptk6-/- mice were injected with azoxymethane alone or in combination with dextran sodium sulfate; formation of aberrant crypt foci and colon tumors was examined. Effects of disruption of Ptk6 on proliferation, apoptosis, and STAT3 activation were examined by immunoblot and immunohistochemical analyses. Regulation of STAT3 activation was examined in the HCT116 colon cancer cell line and young adult mouse colon cells. RESULTS: Ptk6-/- mice developed fewer azoxymethane-induced aberrant crypt foci and tumors. Induction of PTK6 increased apoptosis, proliferation, and STAT3 activation in Ptk6+/+ mice injected with azoxymethane. Disruption of Ptk6 impaired STAT3 activation following azoxymethane injection, and reduced active STAT3 levels in Ptk6-/- tumors. Stable knockdown of PTK6 reduced basal levels of active STAT3, as well as activation of STAT3 by epidermal growth factor in HCT116 cells. Disruption of Ptk6 reduced activity of STAT3 in young adult mouse colon cells. CONCLUSIONS: PTK6 promotes STAT3 activation in the colon following injection of the carcinogen azoxymethane and regulates STAT3 activity in mouse colon tumors and in the HCT116 and young adult mouse colon cell lines. Disruption of Ptk6 decreases azoxymethane-induced colon tumorigenesis in mice.

The alternative splice variant of protein tyrosine kinase 6 negatively regulates growth and enhances PTK6-mediated inhibition of ß-catenin.

Protein tyrosine kinase 6 (PTK6), also called breast tumor kinase (BRK), is expressed in epithelial cells of various tissues including the prostate. Previously it was shown that PTK6 is localized to epithelial cell nuclei in normal prostate, but becomes cytoplasmic in human prostate tumors. PTK6 is also primarily cytoplasmic in the PC3 prostate adenocarcinoma cell line. Sequencing revealed expression of wild type full-length PTK6 transcripts in addition to an alternative transcript lacking exon 2 in PC3 cells. The alternative transcript encodes a 134 amino acid protein, referred to here as ALT-PTK6, which shares the first 77 amino acid residues including the SH3 domain with full length PTK6. RT-PCR was used to show that ALT-PTK6 is coexpressed with full length PTK6 in established human prostate and colon cell lines, as well as in primary cell lines derived from human prostate tissue and tumors. Although interaction between full-length PTK6 and ALT-PTK6 was not detected, ALT-PTK6 associates with the known PTK6 substrates Sam68 and ß-catenin in GST pull-down assays. Coexpression of PTK6 and ALT-PTK6 led to suppression of PTK6 activity and reduced association of PTK6 with tyrosine phosphorylated proteins. While ALT-PTK6 alone did not influence ß-catenin/TCF transcriptional activity in a luciferase reporter assay, it enhanced PTK6-mediated inhibition of ß-catenin/TCF transcription by promoting PTK6 nuclear functions. Ectopic expression of ALT-PTK6 led to reduced expression of the ß-catenin/TCF targets Cyclin D1 and c-Myc in PC3 cells. Expression of tetracycline-inducible ALT-PTK6 blocked the proliferation and colony formation of PC3 cells. Our findings suggest that ALT-PTK6 is able to negatively regulate growth and modulate PTK6 activity, protein-protein associations and/or subcellular localization. Fully understanding functions of ALT-PTK6 and its impact on PTK6 signaling will be critical for development of therapeutic strategies that target PTK6 in cancer.

FoxM1 in tumorigenicity of the neuroblastoma cells and renewal of the neural progenitors.

Malignant neuroblastomas contain stem-like cells. These tumors also overexpress the Forkhead box transcription factor FoxM1. In this study, we investigated the roles of FoxM1 in the tumorigenicity of neuroblastoma. We showed that depletion of FoxM1 inhibits anchorage-independent growth and tumorigenicity in mouse xenografts. Moreover, knockdown of FoxM1 induces differentiation in neuroblastoma cells, suggesting that FoxM1 plays a role in the maintenance of the undifferentiated progenitor population. We showed that inhibition of FoxM1 in malignant neuroblastoma cells leads to the downregulation of the pluripotency genes sex determining region Y box 2 (Sox2) and Bmi1. We provided evidence that FoxM1 directly activates expression of Sox2 in neuroblastoma cells. By using a conditional deletion system and neurosphere cultures, we showed that FoxM1 is important for expression of Sox2 and Bmi1 in the mouse neural stem/progenitor cells and is critical for its self-renewal. Together, our observations suggested that FoxM1 plays an important role in the tumorigenicity of the aggressive neuroblastoma cells through maintenance of the undifferentiated state.

Deregulation of FoxM1b leads to tumour metastasis.

The forkhead box M1b (FoxM1b) transcription factor is over-expressed in human cancers, and its expression often correlates with poor prognosis. Previously, using conditional knockout strains, we showed that FoxM1b is essential for hepatocellular carcinoma (HCC) development. However, over-expression of FoxM1b had only marginal effects on HCC progression. Here we investigated the effect of FoxM1b expression in the absence of its inhibitor Arf. We show that transgenic expression of FoxM1b in an Arf-null background drives hepatic fibrosis and metastasis of HCC. We identify novel mechanisms of FoxM1b that are involved in epithelial-mesenchymal transition, cell motility, invasion and a pre-metastatic niche formation. FoxM1b activates the Akt-Snail1 pathway and stimulates expression of Stathmin, lysyl oxidase, lysyl oxidase like-2 and several other genes involved in metastasis. Furthermore, we show that an Arf-derived peptide, which inhibits FoxM1b, impedes metastasis of the FoxM1b-expressing HCC cells. The observations indicate that FoxM1b is a potent activator of tumour metastasis and that the Arf-mediated inhibition of FoxM1b is a critical mechanism for suppression of tumour metastasis.