Targeting Protein Tyrosine Kinase 6 Enhances Apoptosis of Colon Cancer Cells following DNA Damage

Protein tyrosine kinase 6 (PTK6; also called BRK or Sik) is an intracellular tyrosine kinase distantly related to the Src-family of tyrosine kinases. Although PTK6 is not expressed in the normal mammary gland or ovarian epithelium, it is frequently overexpressed in breast and ovarian tumors (reviewed in refs. 1, 2). In normal tissues, PTK6 is most highly expressed in nondividing, differentiated epithelial cells of the gastrointestinal tract (3–5). PTK6 is also expressed in the normal prostate where it is localized to the epithelial nuclei, but its nuclear localization is lost in prostate disease and prostate tumors (6).

Characterization of the Ptk6-null mouse revealed increased intestinal epithelial cell proliferation and impaired enterocyte differentiation that correlated with enhanced activation of AKT, when compared with wild-type control mice. In addition, nuclear β-catenin was more readily detected in Ptk6-null mice (7). PTK6 can directly associate with β-catenin and inhibit β-catenin/TCF-regulated transcription. Ptk6-null BAT-GAL mice, containing a β-catenin-activated LacZ reporter transgene, displayed increased levels of β-galactosidase expression in the colon (8).

Even though PTK6 promotes epithelial cell differentiation and cell cycle exit in the normal intestine, alterations in PTK6 localization, and/or expression can impact its function. Although PTK6 is primarily restricted to differentiated nondividing cells in the small and large intestine, PTK6 is induced in proliferating progenitor crypt epithelial cells following γ-irradiation (9), or treatment with the carcinogen azoxymethane (AOM; 10). Disruption of the Ptk6 gene impairs DNA damage-induced apoptosis in the mouse. Induction of PTK6 in colonic crypts following AOM injection correlated with increased apoptosis, compensatory proliferation, and tumorigenesis. Reduced tumor development was correlated with impaired STAT3 activation in the colons of Ptk6 null mice (10).

The induction of PTK6 expression following DNA damage in vivo led us to explore potential links between this tyrosine kinase and the tumor suppressor protein p53, which is frequently mutated in colon cancer (11). p53 is a transcription factor that is stabilized following DNA damage. In intestinal tissues, p53-dependent (12, 13) and -independent apoptosis (14) may occur following irradiation. Induction of expression of the cyclin-dependent kinase (CDK) inhibitor p21 may prevent cells from undergoing apoptosis (15), and the ability of p53 to promote expression of p21 has been shown to play a protective role in the intestine (16). Mice lacking either p53 or its downstream target p21 are more sensitive to developing GI toxicity syndrome in response to radiation injury (17).

The aim of our study was to determine if p53 regulates induction of PTK6 expression following DNA damage, and if PTK6 modulates colon cancer cell sensitivity to chemotherapeutic agents. We used HCT116 cells, which were derived from human colorectal carcinoma epithelial cells, and contain a wild-type p53 gene. These cells respond normally to DNA-damaging agents through induction of p53 followed by cell-cycle arrest (18). HCT116 p53^−/− cells were produced by deletion of both alleles of p53 through homologous recombination (19). Using isogenic HCT116 p53^+/+ and p53^−/− cells, we found that knockdown of PTK6 expression enhances apoptosis in p53^+/+ HCT116 colon cancer cells following DNA damage induced by γ-irradiation, doxorubicin, and 5-fluorouracil (5FU). Along with increased apoptosis, PTK6 knockdown cells also displayed decreased survival with impaired STAT3 activation and decreased p21 levels. These data suggest that kinase inhibitors targeting PTK6 may enhance sensitivity of colon cancer cells to chemotherapeutic agents.

The p53^+/+ and p53^−/− HCT116 human colorectal carcinoma cell lines were a gift from Dr. Bert Vogelstein (John Hopkins). Cells were cultured in Dulbecco's Modified Eagle's Medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Immortalized young adult mouse colon (YAMC) control cells were provided by Robert Whitehead (Vanderbilt University Medical Center, Nashville, TN). Control YAMC and YAMC cells derived from Ptk6^−/− mice (20, 21) were cultured in RPMI 1640 media containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and INF-γ (5 U/mL) and grown at 33°C. The Ptk6^+/+ and Ptk6^−/− YAMC cell lines were genotyped and characterized in the authors' laboratory (21). No additional authentication of cell lines was done by the authors.

For γ-irradiation experiments, cells were plated at 4 × 10⁵ cells/10 cm dish 16 hours before treatment. Fresh growth medium was added to the cells and they were exposed to 20 Gy of γ irradiation and harvested at 0, 3, 6, 12, 24, 48, or 72 hours posttreatment. Both floating and attached cells were harvested for protein lysates.

For doxorubicin and 5FU experiments, cells were plated at 1 × 10⁶ cells/10 cm dish 16 hours before treatment. Fresh medium containing either dimethyl sulfoxide (DMSO; control cells), 5 μmol/L or 10 μmol/L of doxorubicin (Fisher Scientific) or 300 μmol/L of 5FU (Sigma-Aldrich) was added per dish, and the cells were incubated for 24 hours at 37°C. Both floating and attached cells were harvested for protein lysates.

The Mission TCR shRNA Target Set directed against PTK6 in the pLKO.1 lentiviral expression vector was purchased from Sigma-Aldrich. Lentiviruses expressing TCRN0000021549 (shRNA 49), TCRN0000021552 (shRNA 52), and empty vector were produced in the HEK293FT packaging cell line by cotransfection with compatible packaging plasmids HIVtrans and vesicular stomatitis virus G protein as described previously (8). Cells were infected with retrovirus (50% viral supernatant and 50% growth medium containing 5 μg/mL polybrene) and placed in selection medium containing 2 μg/mL puromycin for 2 weeks for selection of stable pools.

HCT116 pLKO.1 vector (control) and shRNA49 PTK6 knockdown cells were transfected using the Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions. For ectopic PTK6 overexpression experiments, cells were transfected with DNA encoding full-length wild-type PTK6 (pcDNA3.Myc.PTK6) or empty vector as a control (pcDNA3). The cells were plated in 10 cm dishes, transfected with 8 μg DNA and incubated at 37°C for 12 hours before treatment with doxorubicin or 5FU.

Immunoblotting was conducted as previously described (7). Antibodies were obtained from the following sources: human PTK6, Millipore; AKT (9272), phospho-AKT (Thr308), cleaved-caspase-3, cleaved PARP, ERK1/2 (9102), phospho-ERK1/2 (Thr202/Tyr204), ERK5 (3372), phospho-ERK5 (Thr218/Tyr220), STAT3 (9132) and phospho-STAT3 (Tyr705), Cell Signaling Technology; p53 (DO-1) HRP, Santa Cruz Biotechnology, Inc; β-actin, Sigma-Aldrich; and p21, BD Biosciences. Secondary antibodies (donkey anti-rabbit or sheep anti-mouse conjugated to horseradish peroxidase; Amersham Biosciences) were detected by chemiluminescence with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific).

The Annexin V-PE Staining Kit (Enzo Life Sciences) was used for the detection of apoptotic cells, according to manufacturer's instructions. This kit uses a dual-staining protocol in which the cells show fluorescence of both Annexin V (apoptotic cells) 7AAD (necrotic cells or late apoptotic cells). HCT116 cells were exposed to 20 Gy of γ-irradiation and harvested 48 hours posttreatment. Cells were trypsinized and 1 × 10⁵ cells were washed with PBS, processed for labeling with Annexin V-7AAD, and analyzed by flow cytometry.

Cells were seeded at a density of 1 × 10⁵ cells/plate on 10 cm dishes. Control (nonirradiated) or irradiated cells exposed to 20 Gy of γ-irradiation were grown for 7 days. The cells were stained with crystal violet solution (0.1% crystal violet and 25% methanol in water) for 30 minutes at room temperature.

Total RNA was isolated from cells using TRIzol (Invitrogen) according to manufacture's instructions. To generate cDNA for real-time PCR (RT-PCR), total RNA was reverse transcribed using the SuperScript III First-Strand kit (Invitrogen). Quantitative RT-PCR (qRT-PCR) was conducted using the MyiQ single-color real-time PCR detection system (Bio-Rad). RT-PCR amplification was always done in triplicate and a melting curve was conducted for each analysis to ensure the amplification of one product. The levels of each gene of interest were normalized against the levels of 18S mRNA, which was used as an internal control. The following primers were used: human p21 (Sense, 5′-AAG ACC ATG TGG ACC TGT -3′; Antisense, 5′- GGT AGA AAT CTG TCA TGC TG -3′), human PTK6 (Sense, 5′-TGT TCC TGC TCT TCC CAG TT-3′; Antisense, 5′- TGG GAG GAA AGA ACC CTT GA-3′; ref. 22), and human 18S (Sense, 5′-TTG ACT CAA CAC GGG AAA CC-3′; Antisense, 5′-ACC CAC GGA ATC GAG AAA GA-3′).

Results are shown as the mean ± SD. P values were determined using the 2-tailed Student t test (Microsoft Excel, 2010). A difference was considered statistically significant if the P value was equal to or less than 0.05. Immunoblot band densities were quantified using NIH ImageJ (23).

Following DNA damage, PTK6 is induced in the proliferating crypt epithelial cells in the small intestine (9) and colon (10), where it promotes apoptosis. Apoptosis in the intestine following γ-irradiation is regulated by p53-dependent (12, 13) and p53-independent (14) mechanisms. To determine if p53 regulates induction of PTK6 expression following DNA damage, we turned to the isogenic HCT116 p53^+/+ and p53^−/− human colon cancer cell lines (24) that express moderate levels of endogenous PTK6. Both cell lines were subjected to 20 Gy of γ-irradiation and harvested at 0, 6, 24, 48, and 72 hours posttreatment. Protein lysates were prepared and analyzed by immunoblotting. Induction of PTK6 was observed by 24 hours after irradiation, with peak levels at 48 hours (Fig. 1A and B). Although PTK6 was induced in both HCT116 p53^+/+ and p53^−/− cell lines following irradiation, significantly higher levels of PTK6 protein were detected in p53^+/+ HCT116 cells (Fig. 1A and B) suggesting a p53-dependent component in PTK6 induction. Rapid accumulation of p53 and activation of the p53 target gene p21 were detected in wild-type HCT116 cells following irradiation (Fig. 1A).

To further explore links between p53 and PTK6 induction, expression of PTK6 and the p53 target gene p21 were examined by qRT-PCR. HCT116 p53^+/+ and p53^−/− cell lines were subjected to 20 Gy of γ-irradiation and harvested at 0, 3, 6, 24, 48, and 72 hours posttreatment. Induction of PTK6 mRNA expression was observed, with an approximately 30-fold increase in expression at 48 hours postirradiation in the HCT116 p53^+/+ cells (Fig. 1C). However, the p21 gene, which is a direct target of p53 (25), showed a 90-fold induction by 3 hours postirradiation in HCT116 p53^+/+ cells (Fig. 1D). Induction of PTK6 mRNA is consistent with the induction of PTK6 protein expression at 48 hours postirradiation, but unlike the p21 gene that is induced by 3 hours postirradiation, the PTK6 gene does not appear to be a direct transcriptional target of p53.

DNA-damaging agents including γ-irradiation and chemotherapeutic drugs, such as doxorubicin and 5FU, are the basis of most current cancer treatment regimens. Given the important role of p53 in DNA damage-induced apoptosis, we wanted to assess the potential cross talk between p53 and PTK6 in regulating DNA damage-induced apoptosis following γ-irradiation. HCT116 p53^+/+ and p53^−/− cell lines containing empty vector (V) or 1 of 2 different short hairpin RNAs (shRNA; refs. 49, 52) that target PTK6 were subjected to 20 Gy of γ-irradiation and harvested at 0, 6, and 48 hours posttreatment. DNA damage-induced expression of p53 was detected in HCT116 p53^+/+ vector control and stable PTK6 knockdown cell lines by 6 hours (Fig. 2A). In addition, p21 expression was induced following γ-irradiation in both HCT116 p53^+/+ and p53^−/− cells, but reduced p21 expression was observed in PTK6 knockdown cells (49, 52) versus control cells (V; Fig. 2B). These results support the findings that PTK6 expression enhances p21 expression, possibly leading to survival of colon cancer cells following DNA damage. Immunoblotting with antibodies specific for cleaved caspase-3 and cleaved PARP further shows that knockdown of PTK6 leads to enhanced apoptosis in the p53^+/+ HCT116 cells (Fig. 2C).

The PTK6 knockdown cells were subjected to γ-irradiation and incubated with PE Annexin V and 7AAD, and cell death was analyzed by flow cytometry. PE Annexin V staining can identify early stages of apoptosis, whereas staining cells with 7AAD detects the late stages of cell death resulting from either apoptosis or necrosis. Both HCT116 p53^+/+ and p53^−/− cells displayed increased apoptosis following irradiation (Fig. 3A and B). Stable knockdown of PTK6 in HCT116 p53^+/+ cells led to increased apoptosis compared with vector control cells (Fig. 3A), whereas PTK6 expression had little to no effect on apoptosis in HCT 116 p53^−/− cells following irradiation (Fig. 3B). To further examine the impact that knockdown of PTK6 had on cell growth and survival, we conducted colony assays with HCT116 p53^+/+ and p53^−/− PTK6 knockdown cells following 20 Gy of γ-irradiation. Colony formation was assessed at 7 days postirradiation. Although 20 Gy of γ-irradiation leads to dramatic induction of cell death in both HCT116 p53^+/+ and p53^−/− cells, a population of cells survived and gave rise to colonies (Fig. 3C). Both HCT116 p53^+/+ and p53^−/− cells displayed decreased survival following γ-irradiation with a more dramatic decrease observed in HCT116 p53^−/− cells (Fig. 3C). Stable knockdown of PTK6 led to a more striking decrease in survival in HCT116 p53^+/+ cells compared with vector control cells.

Doxorubicin is a DNA-damaging anthracycline antibiotic used in chemotherapy treatment of a wide range of malignancies (26). The chemotherapeutic agent 5FU is one of the most effective adjuvant therapies for patients with colon cancer (27). Both agents induce apoptosis in sensitive cancer cells. To determine if PTK6 modulates colon cancer cell sensitivity to chemotherapeutic agents, HCT116 p53^+/+ and p53^−/− cells expressing vector control (V) or shRNAs targeting PTK6 (49 and 52) were treated with doxorubicin or 5FU for 24 hours, and cell lysates were prepared and examined by immunoblotting. Induction of PTK6 was observed following treatment with doxorubicin (Fig. 4A) with a more marked increase detected in HCT116 p53^+/+ cells as compared with p53^−/− cells. In HCT116 p53^+/+ cells treated with doxorubicin, the highest levels of apoptosis, marked by cleaved caspase-3 and cleaved PARP levels, were detected in PTK6 knockdown cells compared with vector control cells (Fig. 4A). PTK6 expression had little effect on apoptosis in HCT116 p53^−/− cells following drug treatment. Induction of p21 was also detected in HCT116 p53^+/+ cells following treatment, with significantly higher levels observed in vector control cells compared with PTK6 knockdown cells (Fig. 4A). A slight induction of p21 was observed in HCT116 p53^−/− cells following 10 μmol/L of doxorubicin indicating a p53-independent mode of regulation. However, PTK6 knockdown had no significant impact on p21 induction in HCT116 p53^−/− cells following doxorubicin treatment.

The effect of 5FU on apoptosis has been attributed to the ability of this drug to induce the activity of p53. Mutation or deletion of p53 has been shown to lead to resistance of cells to 5FU (19, 27). HCT116 p53^+/+ cells treated with 5FU show a similar trend to that of doxorubicin with both induction of PTK6 expression and enhanced apoptosis in PTK6 knockdown cells (Fig. 4B). 5FU treatment did not cause detectable apoptosis in the HCT116 p53^−/− cells (Fig. 4B). Induction of p21 was also detected in HCT116 p53^+/+ cells following 5FU treatment, with significantly higher levels observed in vector control cells compared with PTK6 knockdown cells (Fig. 3B). Little to no expression of p21 was observed in HCT116 p53^−/− cells following drug treatment, as p21 induction by DNA-damaging agents is primarily regulated by p53 (28). These data suggest that knockdown of PTK6 expression promotes apoptosis of HCT116 p53^+/+ cells and enhances the response of colon tumor cells to doxorubicin and 5FU.

To verify that the enhanced apoptosis in PTK6 knockdown cells was not because of off-target shRNA effects, we reintroduced recombinant PTK6 into these cells. The shRNA49 targets the 3′ untranslated region of Ptk6, which is not present in the recombinant construct, so we used the shRNA49 and pLKO.1 vector control stable cell lines for ectopic PTK6 expression (PTK6 shRNA, pLKO.1; Fig. 5A). Following transient transfection of wild-type recombinant PTK6 or empty vector control (pcDNA3), cells were treated with doxorubicin or 5FU for 24 hours, and cell lysates were prepared and examined by immunoblotting (Fig. 5B). In PTK6 knockdown cells (PTK6 shRNA), doxorubicin- and 5FU-induced cleavage of caspase-3 and PARP were significantly reduced by ectopic wild-type PTK6 (Fig. 5C). Rescue of apoptosis by expression of ectopic PTK6 confirms that the enhanced apoptosis of PTK6 knockdown cells in response to DNA damage was because of reduced PTK6 expression in those cells.

To identify signaling pathways downstream of PTK6 that might play a role in cell survival, we examined the expression and activation of, AKT, ERK1/2, ERK5, and STAT3. Previously, we showed that AKT and ERK1/2 activation was increased following disruption of PTK6 in the mouse small intestine following γ-irradiation (9). To determine if these prosurvival pathways were affected, HCT116 p53^+/+ cells with stable knockdown of PTK6 and control cells were treated with 5 μmol/L or 10 μmol/L of doxorubicin for 24 hours and activation of AKT and ERK1/2 was examined using phospho-specific antibodies. We detected increased activation of all signaling proteins following DNA damage, but knockdown of PTK6 had no impact on activation (Fig. 6A). We also examined ERK5 activation as it has been shown to play a role in cancer cell survival and proliferation (29). PTK6, in complex with ERK5, has been shown to regulate cell migration in keratinocytes and breast cancer cells following receptor activation (30, 31). Similar to AKT and ERK1/2, we observed an increase in phospho-ERK5 levels following treatment, but PTK6 expression had no effect on ERK5 activation (Fig. 6A).

STAT3 has been shown to promote proliferation and survival leading to colon tumorigenesis (32, 33). PTK6 phosphorylates and activates STAT3 in cell lines (34) and increased STAT3 activation was observed in Ptk6^+/+ mice, compared with Ptk6^−/− mice, after AOM administration (10). We examined STAT3 activation in cell lysates prepared from vector control (V) and PTK6 stable knockdown HCT116 p53^+/+ cells (49 and 52) that were treated with 5 μmol/L or 10 μmol/L of doxorubicin for 24. We examined STAT3 activation by examining phosphorylation of STAT3 tyrosine residue 705 (P-STAT3). Highest levels of P-STAT were detected in the vector control cells. Following doxorubicin treatment, there was an increase in STAT3 activation in PTK6 knockdown cells (49, 52) but not to the levels observed in vector control cells (Fig. 6B).

We also examined STAT3 activation in immortalized YAMC cells from wild-type and Ptk6^−/− mice (21). This cell culture model system represents normal colon epithelial cells (21). Lysates were prepared from untreated or γ-irradiated Ptk6^+/+ and Ptk6^−/− YAMC cells. Interestingly, both p53 and p21 were induced in irradiated control and Ptk6 null YAMC cells, which carry a temperature-sensitive mutant of the SV40 large T gene for immortalization and have impaired p53 activity. In Ptk6^−/− YAMC cells, p21 levels were slightly lower than in wild-type control YAMCs postirradiation. Previously, we showed that PTK6 regulates basal STAT3 activity in YAMC cells (10). Similar results were obtained from YAMC cells following γ-irradiation treatment. Baseline levels of phospho-STAT3 are higher in Ptk6^+/+ YAMC cells compared with Ptk6^−/− YAMC cells. In cells that were exposed to 20 Gy of γ-irradiation and harvested at 48 hours posttreatment, active STAT3 was detected only in Ptk6^+/+ cells (Fig. 6C). Consistent with the HCT116 cells, we observed increased cleaved caspase-3 levels in Ptk6^−/− YAMC cells compared with the wild-type cells following radiation. These data suggest that the protective role of PTK6 in response to DNA damage may function through activation of the prosurvival transcription factor STAT3.

Functions of PTK6 are not well understood and a growing body of evidence indicates that PTK6 signaling outcomes are context-dependent. PTK6 has growth-suppressive or growth-promoting functions dependent on its localization in different intracellular compartments (8, 35–39). Although it is not expressed in normal mammary gland, high level expression of PTK6 in a majority of breast tumors examined may promote ERBB family, MET (reviewed in ref. 2), and insulin-like growth factor-1 receptor signaling (40). Studies of PTK6 in the normal intestine indicated that PTK6 restrained growth and promoted differentiation of gut epithelial cells (7). However, PTK6 induction in progenitor cells following DNA damage led to inhibition of prosurvival signaling and apoptosis (9). Although characterization of PTK6 in the normal gastrointestinal tract suggested that it might function as a tumor suppressor, recent in vivo studies have shown that disruption of Ptk6 impairs STAT3 activation and subsequent tumorigenesis following carcinogen administration (10).

These studies are the first to link PTK6 with the tumor suppressor p53 in intestinal cells. Radiation and chemotherapeutic agents stabilize and activate p53, which regulates genes that encode proteins that function in cell-cycle regulation, survival, and apoptosis (41). Recently, p53 has been implicated in preventing the epithelial–mesenchymal transition (42). We show that PTK6 expression is positively regulated by p53-dependent mechanisms following DNA damage in HCT116 cells. We found that PTK6 protein and mRNA levels were induced in HCT116 p53^+/+ cells following γ-irradiation (Fig. 1A–C). However, unlike the p21 gene that is induced by 3 hours post-γ-irradiation, the PTK6 gene, which is induced at peak levels at 48 hours post-γ-irradiation, does not appear to be a direct target of p53 (Fig. 1C and D).

Our data indicate that PTK6 expression confers resistance of colon cancer cells to DNA damaging agents, such as doxorubicin and 5FU and γ-radiation. Unlike what we observed in the small intestine (9) and colon (10) in vivo, knockdown of PTK6 in HCT116 p53^+/+ and p53^−/− cells led to enhanced apoptosis in HCT116 p53^+/+ cells following γ-radiation, doxorubicin, and 5FU treatment. Increased apoptosis was also observed in YAMC Ptk6^−/− cells (Fig. 6C). These data suggest that knockdown of PTK6 in colon cancer cells will enhance sensitivity to DNA damage-induced apoptosis.

One of the best-characterized p53 targets, the CDK inhibitor p21, is expressed in differentiated intestinal epithelial cells similar to PTK6 (43). In several systems, p21 has been shown to promote cell survival by inducing cell-cycle arrest and DNA repair (reviewed in refs. 15, 44). The ability of p53 to activate p21 has a protective effect in the intestinal epithelium following high doses of radiation (17, 45, 46). Expression of p21 has been correlated with the resistance of human colon cancer cell lines to chemotherapeutic agents (19, 47). Knockdown of PTK6 in HCT116 p53^+/+ cells led to decreased p21 expression following treatment with γ-irradiation, doxorubicin, and 5FU (Figs. 2 and 3). These data suggest a novel cross talk between p53 and PTK6 that merits further exploration. The ability of PTK6 to contribute to p21 expression following DNA damage provides one mechanism by which it may promote cell survival.

Along with an increase in apoptosis, PTK6 knockdown cells displayed decreased survival following γ-irradiation in the HCT116 p53^+/+ cells (Fig. 3C). STAT3 activation has been shown to play a vital role in a variety of cellular processes including proliferation, migration, and survival (48). In the gastrointestinal tract, STAT3 has been shown to play a critical role in the initiation and progression of colitis-associated colon cancer (32, 33). STAT3 has been shown to play an important role in intestinal stem cell survival (49), and it will be interesting to determine contributions of STAT3 and PTK6 to putative colon cancer stem cells. STAT3 was identified as a substrate of PTK6 (34), and we determined that PTK6 regulates activation of STAT3 in the colon after AOM-induced DNA damage, in established tumors and in the HCT116 colon cancer cell line (10). Consistent with previous results, STAT3 activation was impaired in HCT116 p53^+/+ cells following doxorubicin treatment (Fig. 6B) and in YAMC Ptk6 knockout cells after γ-irradiation (Fig. 6C). The ability of PTK6 to regulate STAT3 activity could contribute to resistance of colon cancer cells to DNA damaging agents.

In a complex tissue such as the colon, epithelial cells located on different positions along the crypt-villus axis are subjected to different signals emanating from the environment. When PTK6 is induced in crypt epithelial cells following DNA damage, it contributes to the apoptotic response (9, 10). In contrast, PTK6 promotes resistance to DNA damage-induced apoptosis in human colon cancer cells. Development of colon cancers is accompanied by mutations and alterations in a number of genes and pathways, changing the cellular context for PTK6 signaling. Knockdown of PTK6 following DNA damage led to reduced p21 expression and STAT3 activity, leading to enhanced apoptosis and decreased survival. These data suggest that identification of kinase inhibitors that specifically target PTK6 may enhance sensitivity of colon cancer cells to chemotherapeutic agents and radiation.

No potential conflicts of interest were disclosed by the other authors.

Conception and design: J.J. Gierut, A.L. Tyner

Development of methodology: J.J. Gierut

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.J. Gierut, P.S. Mathur, W. Bie, J. Han

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.J. Gierut, P.S. Mathur, J. Han, A.L. Tyner

Writing, review, and/or revision of the manuscript: J.J. Gierut, P.S. Mathur, A.L. Tyner

Study supervision: A.L. Tyner

The authors thank the laboratories of Dr. Bert Vogelstein (Johns Hopkins University) and Dr. Robert Whitehead (Vanderbilt University) for providing cell lines for the studies.

This work was supported by NIH Grant DK44525 (A.L. Tyner). J.J. Gierut received support from a NRSA/NIH Institutional T32 training grant DK07739, and an AGA Foundation Graduate Student Research Fellowship Award.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.