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.

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.

FoxM1, a critical regulator of oxidative stress during oncogenesis.

The transcription factor FoxM1 is over-expressed in most human malignancies. Although it is evident that FoxM1 has critical functions in tumour development and progression, the mechanisms by which FoxM1 participates in those processes are not understood. Here, we describe an essential role of FoxM1 in the regulation of oxidative stress that contributes to malignant transformation and tumour cell survival. We identify a negative feedback loop involving FoxM1 that regulates reactive oxygen species (ROS) in proliferating cells. We show that induction of FoxM1 by oncogenic Ras requires ROS. Elevated FoxM1, in turn, downregulates ROS levels by stimulating expression of ROS scavenger genes, such as MnSOD, catalase and PRDX3. FoxM1 depletion sensitizes cells to oxidative stress and increases oncogene-induced premature senescence. Moreover, tumour cells expressing activated AKT1 are 'addicted' to FoxM1, as they require continuous presence of FoxM1 for survival. Together, our results identify FoxM1 as a key regulator of ROS in dividing cells, and provide insights into the mechanism how tumour cells use FoxM1 to control oxidative stress to escape premature senescence and apoptosis.

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.

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.

Identification of beta-catenin as a target of the intracellular tyrosine kinase PTK6.

Disruption of the gene encoding protein tyrosine kinase 6 (PTK6) leads to increased growth, impaired enterocyte differentiation and higher levels of nuclear beta-catenin in the mouse small intestine. Here, we demonstrate that PTK6 associates with nuclear and cytoplasmic beta-catenin and inhibits beta-catenin- and T-cell factor (TCF)-mediated transcription. PTK6 directly phosphorylates beta-catenin on Tyr64, Tyr142, Tyr331 and/or Tyr333, with the predominant site being Tyr64. However, mutation of these sites does not abrogate the ability of PTK6 to inhibit beta-catenin transcriptional activity. Outcomes of PTK6-mediated regulation appear to be dependent on its intracellular localization. In the SW620 colorectal adenocarcinoma cell line, nuclear-targeted PTK6 negatively regulates endogenous beta-catenin/TCF transcriptional activity, whereas membrane-targeted PTK6 enhances beta-catenin/TCF regulated transcription. Levels of TCF4 and the transcriptional co-repressor TLE/Groucho increase in SW620 cells expressing nuclear-targeted PTK6. Knockdown of PTK6 in SW620 cells leads to increased beta-catenin/TCF transcriptional activity and increased expression of beta-catenin/TCF target genes Myc and Survivin. Ptk6-null BAT-GAL mice, containing a beta-catenin-activated LacZ reporter transgene, have increased levels of beta-galactosidase expression in the gastrointestinal tract. The ability of PTK6 to negatively regulate beta-catenin/TCF transcription by modulating levels of TCF4 and TLE/Groucho could contribute to its growth-inhibitory activities in vivo.

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.

Building a better understanding of the intracellular tyrosine kinase PTK6 - BRK by BRK.

Protein tyrosine kinase 6 (PTK6), also referred to as breast tumor kinase BRK, is a member of a distinct family of kinases that is evolutionarily related to the SRC family of tyrosine kinases. While not expressed in the normal mammary gland, PTK6 expression is detected in a large proportion of human mammary gland tumors. In breast tumor cells, PTK6 promotes growth factor signaling and cell migration. PTK6 expression is also increased in a number of other epithelial tumors, including ovarian and colon cancer. In contrast, PTK6 is expressed in diverse normal epithelia, including the linings of the gastrointestinal tract, skin and prostate, where its expression correlates with cell cycle exit and differentiation. Disruption of the mouse Ptk6 gene leads to increased growth and impaired differentiation in the small intestine that is accompanied by increased AKT and Wnt signaling. Following total body irradiation, PTK6 expression is induced in proliferating progenitor cells of the intestine, where it plays an essential role in DNA-damage induced apoptosis. A distinguishing feature of PTK6 is its flexibility in intracellular localization, due to a lack of amino-terminal myristoylation/palmitoylation. Recently a number of substrates of PTK6 have been identified, including nuclear RNA-binding proteins and transcription factors. We discuss PTK6 signaling, its apparent conflicting roles in cancer and normal epithelia, and its potential as a therapeutic target in epithelial cancers.

A conserved phosphorylation site within the forkhead domain of FoxM1B is required for its activation by cyclin-CDK1.

The Forkhead box M1 (FoxM1) transcription factor is critical for expression of the genes essential for G(1)/S transition and mitotic progression. To explore the cell cycle regulation of FoxM1, we examined the phosphorylation profile of FoxM1. Here, we show that the phosphorylated status and the activity of FoxM1 increase as cells progress from S to G(2)/M phases. Moreover, dephosphorylation of FoxM1 coincides with exit from mitosis. Using mass spectrometry, we have identified a new conserved phosphorylation site (Ser-251) within the forkhead domain of FoxM1. Disruption of Ser-251 inhibits phosphorylation of FoxM1 and dramatically decreases its transcriptional activity. We demonstrate that the Ser-251 residue is required for CDK1-dependent phosphorylation of FoxM1 as well as its interaction with the coactivator CREB-binding protein (CBP). Interestingly, the transcriptional activity of the S251A mutant protein remains responsive to activation by overexpressed Polo-like kinase 1 (PLK1). Cells expressing the S251A mutant exhibit reduced expression of the G(2)/M phase genes and impaired mitotic progression. Our results demonstrate that the transcriptional activity of FoxM1 is controlled in a cell cycle-dependent fashion by temporally regulated phosphorylation and dephosphorylation events, and that the phosphorylation at Ser-251 is critical for the activation of FoxM1.

Induction of protein tyrosine kinase 6 in mouse intestinal crypt epithelial cells promotes DNA damage-induced apoptosis.

BACKGROUND & AIMS: Protein tyrosine kinase 6 (PTK6) is expressed in epithelial linings of the gastrointestinal tract. PTK6 sensitizes the nontransformed Rat1a fibroblast cell line to apoptotic stimuli. The aim of this study was to determine if PTK6 regulates apoptosis in vivo after DNA damage in the small intestine. METHODS: Wild-type and Ptk6(-/-) mice were subjected to gamma-irradiation; intestinal tissues were collected, protein was isolated, and samples were fixed for immunohistochemical analyses at 0, 6, and 72 hours after the mice were irradiated. Expression of PTK6 was examined in the small intestine before and after irradiation. Apoptosis and proliferation were compared between wild-type and Ptk6(-/-) mice. Expression and activation of prosurvival signaling proteins were assessed. RESULTS: Irradiation induced PTK6 in crypt epithelial cells of the small intestine in wild-type mice. Induction of PTK6 corresponded with DNA damage-induced apoptosis in the wild-type small intestine. Following irradiation, the apoptotic response was impaired in the intestinal crypts of Ptk6(-/-) mice. Increased activation of AKT and extracellular signal-regulated kinase (ERK)1/2 and increased inhibitory phosphorylation of the proapoptotic protein BAD were detected in Ptk6(-/-) mice after irradiation. In response to the induction of apoptosis, compensatory proliferation increased in the small intestines of wild-type mice but not in Ptk6(-/-) mice at 6 hours after irradiation. CONCLUSIONS: PTK6 is a stress-induced kinase that promotes apoptosis by inhibiting prosurvival signaling. After DNA damage, induction of PTK6 is required for efficient apoptosis and inhibition of AKT and ERK1/2.

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.

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 negatively regulates growth and promotes enterocyte differentiation in the small intestine.

Protein tyrosine kinase 6 (PTK6) (also called Brk or Sik) is an intracellular tyrosine kinase that is expressed in breast cancer and normal epithelial linings. In adult mice, PTK6 expression is high in villus epithelial cells of the small intestine. To explore functions of PTK6, we disrupted the mouse Ptk6 gene. We detected longer villi, an expanded zone of PCNA expression, and increased bromodeoxyuridine incorporation in the PTK6-deficient small intestine. Although differentiation of major epithelial cell types occurred, there was a marked delay in expression of intestinal fatty acid binding protein, suggesting a role for PTK6 in enterocyte differentiation. However, fat absorption was comparable in wild-type and Ptk6-/- mice. It was previously shown that the serine threonine kinase Akt is a substrate of PTK6 and that PTK6-mediated phosphorylation of Akt on tyrosine resulted in inhibition of Akt activity. Consistent with these findings, we detected increased Akt activity and nuclear beta-catenin in intestines of PTK6-deficient mice and decreased nuclear localization of the Akt substrate FoxO1 in villus epithelial cells. PTK6 contributes to maintenance of tissue homeostasis through negative regulation of Akt in the small intestine and is associated with cell cycle exit and differentiation in normal intestinal epithelial cells.

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.

Conditionally immortalized colonic epithelial cell line from a Ptk6 null mouse that polarizes and differentiates in vitro.

BACKGROUND AND AIMS: PTK6 is an intracellular src-related tyrosine kinase that regulates differentiation in the intestine, where knockout animals have increased proliferative activity and growth characteristics. To explore the phenotype further we attempted to establish epithelial cell lines from the intestinal mucosa. METHOD: We mated Ptk6 null mice with a tsSV40 large T transgenic mouse (Immortomouse) to obtain null mice carrying the SV40 gene. Intestinal tissues from these mice were cultured. RESULTS: We established a Ptk6 null epithelial cell line from the colonic mucosa. Consistent with a role of Ptk6 in cell differentiation, these cells have the characteristics of a stable progenitor cell. In monolayer culture, the cells form domes in the monolayer when confluent. When cultured on Transwell filters, the cells polarize and form an electrically resistant barrier. Formation of tight junctions was confirmed by demonstrating expression of ZO1 and occludin at the apical junctions, whereas E-cadherin localized to the basolateral membrane. When cultured in collagen gel, the Ptk6 null cells form complex organoids, some of which resemble cups of cells. These organoids contain cells with differentiated phenotypes. Using immunohistochemistry and confocal microscopy we have been able to identify villin-positive (absorptive cells) and a small percentage of mucin-containing cells (goblet cells) and chromogranin A-positive cells (endocrine cells). CONCLUSION: This conditionally immortalized cell line represents an excellent cell culture model system for exploring the mechanisms of cell function and epithelial differentiation in the colonic mucosa.

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.

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.

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.

Altered localization and activity of the intracellular tyrosine kinase BRK/Sik in prostate tumor cells.

Breast tumor kinase (BRK) is an intracellular tyrosine kinase expressed in differentiating epithelial cells of the gastrointestinal tract and skin, and in several epithelial cancers including carcinomas of the breast and colon. We examined expression of BRK and its mouse ortholog Src-related intestinal kinase (Sik) in prostate tissues and detected it in the nuclei of normal luminal prostate epithelial cells. BRK localization was then examined in 58 human prostate biopsy samples representing various grades of prostate cancer. While nuclear localization of BRK was present in well-differentiated tumors, it was absent in poorly differentiated tumors. However localization of Sam68, a nuclear substrate of BRK/Sik, was unaltered in all prostate tumors examined. Consistent with these results, nuclear BRK was detected in the more differentiated androgen-responsive LNCaP human prostate cancer cell line that is poorly tumorigenic in host animals, but it was primarily cytoplasmic in the undifferentiated androgen-unresponsive PC3 prostate cancer cell line that forms aggressive tumors. While PC3 cells expressed higher levels of endogenous BRK than LNCaP cells, BRK was less active in these cells. Our data suggest that BRK plays a role in differentiation of prostate epithelial cells. Altered BRK localization and/or activity may provide a prognostic indicator for prostate tumor progression and be a potential target for therapeutic intervention.

Tumor suppressor functions for the Cdk inhibitor p21 in the mouse colon.

The Cdk inhibitor p21 regulates p53-mediated growth arrest following DNA damage. It is expressed during epithelial differentiation in a variety of organs including colon. We investigated susceptibility of p21-deficient mice to the colon carcinogen azoxymethane (AOM). After AOM injections, rodents develop putative premalignant lesions called aberrant crypt foci (ACF) that are localized to the distal three centimeters of the colon. p21-deficient mice developed significantly higher numbers of ACF than wild-type mice in response to AOM, and these were not restricted to distal colon. After AOM treatment, increased numbers of lymphoid aggregates were detected in p21-deficient colon. Proliferation was similar in wild type and p21-deficient colon before and after AOM injection, but AOM-induced apoptosis was detected only in wild-type crypt epithelial cells, and not in the p21-deficient colon. The proapoptotic function uncovered for p21 was unexpected, because p21 acts as an inhibitor of apoptosis in many systems, and is not required for p53-dependent apoptosis. Enhanced formation of ACF in p21-deficient mice supports a tumor suppressor function for p21 in the colon. Reduced apoptosis of colon epithelial cells with deleterious mutations may be an initiating event in the formation of ACF, with inflammatory cell cytokine expression contributing to their further expansion.