BACKGROUND: BRK is, a non-receptor tyrosine kinase, overexpressed in approximately 85% of human invasive ductal breast tumors. It is not clear whether BRK expression correlates with breast cancer subtypes, or the expression has prognostic or diagnostic significance. Herein, we investigated the correlation of BRK with any breast cancer subtypes and clinicopathological significance of BRK expression in breast cancer.
METHODS: In this study, we examined BRK expression in 120 breast tumor samples and 29 breast cancer cell lines to explore the positive correlation between BRK and the expression of ERα. We used immunohistochemistry, RT-PCR, and immunoblotting to analyse our experimental samples.
RESULT: We demonstrate that estrogen induces BRK gene and protein expression in ER+ breast cancer cells. Over-expression of ERα in the ER-negative breast cancer cell line increased BRK expression, and knock-down of ESR1 in MCF7 cells reduced BRK levels. Further, we provide evidence that BRK is regulated by ERα signaling and the presence of ER antagonists (tamoxifen and fulvestrant) reduce the expression of BRK in ER-positive breast cancer cells. Finally, we demonstrate that the overall survival of ER-positive breast cancer patients is poor when their cancers express high levels of BRK.
CONCLUSION: Our data indicate that BRK is a prognostic marker for ER+ breast cancers and provide a strong rationale for targeting BRK to improve patients' survival.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is one mechanism of carcinoma migration, while complex tumour migration or bulk migration is another - best demontrated by tumour cells invading blood vessels.
METHODS: Thirty cases of non-small cell lung carcinomas were used for identifying genes responsible for bulk cell migration, 232 squamous cell and adenocarcinomas to identify bulk migration rates. Genes expressed differently in the primary tumour and in the invasion front were regarded as relevant in migration and further validated in 528 NSCLC cases represented on tissue microarrays (TMAs) and metastasis TMAs.
RESULTS: Markers relevant for bulk cancer cell migration were regulated differently when compared with EMT: Twist expressed in primary tumour, invasion front, and metastasis was not associated with TGFβ1 and canonical Wnt, as Slug, Snail, and Smads were negative and β-Catenin expressed membraneously. In the majority of tumours, E-Cadherin was downregulated at the invasive front, but not absent, but, coexpressed with N-Cadherin. Vimentin was coexpressed with cytokeratins at the invasion site in few cases, whereas fascin expression was seen in a majority. Expression of ERK1/2 was downregulated, PLCγ was only expressed at the invasive front and in metastasis. Brk and Mad, genes identified in Drosophila border cell migration, might be important for bulk migration and metastasis, together with invadipodia proteins Tks5 and Rab40B, which were only upregulated at the invasive front and in metastasis. CXCR1 was expressed equally in all carcinomas, as opposed to CXCR2 and 4, which were only expressed in few tumours.
CONCLUSION: Bulk cancer cell migration seems predominant in AC and SCC. Twist, vimentin, fascin, Mad, Brk, Tsk5, Rab40B, ERK1/2 and PLCγ are associated with bulk cancer cell migration. This type of migration requires an orchestrated activation of proteins to keep the cells bound to each other and to coordinate movement. This hypothesis needs to be proven experimentally.

Protein tyrosine kinase 6 (PTK6, or BRK) is aberrantly expressed in breast cancers, and emerging as an oncogene that promotes tumor cell proliferation, migration and evasion. Both kinase-dependent and -independent functions of PTK6 in driving tumor growth have been described, therefore targeting PTK6 kinase activity by small molecule inhibitors as a therapeutic approach to treat cancers remains to be validated. In this study, we identified novel, potent and selective PTK6 kinase inhibitors as a means to investigate the role of PTK6 kinase activity in breast tumorigenesis. We report here the crystal structures of apo-PTK6 and inhibitor-bound PTK6 complexes, providing the structural basis for small molecule interaction with PTK6. The kinase inhibitors moderately suppress tumor cell growth in 2D and 3D cell cultures. However, the tumor cell growth inhibition shows neither correlation with the PTK6 kinase activity inhibition, nor the total or activated PTK6 protein levels in tumor cells, suggesting that the tumor cell growth is independent of PTK6 kinase activity. Furthermore, in engineered breast tumor cells overexpressing PTK6, the inhibition of PTK6 kinase activity does not parallel the inhibition of tumor cell growth with a >500-fold shift in compound potencies (IC50 values). Overall, these findings suggest that the kinase activity of PTK6 does not play a significant role in tumorigenesis, thus providing important evidence against PTK6 kinase as a potential therapeutic target for breast cancer treatment.

The triple-negative breast cancer subtype is highly aggressive and has no defined therapeutic target. Fyn-related kinase (FRK) is a non-receptor tyrosine kinase, reported to be downregulated in breast cancer and gliomas, where it is suggested to have tumor suppressor activity. We examined the expression profile of FRK in a panel of 40 breast cancer cells representing all the major subtypes, as well as in 4 non-malignant mammary epithelial cell lines. We found that FRK expression was significantly repressed in a proportion of basal B breast cancer cell lines. We then determined the mechanism of suppression of FRK in FRK-low or negative cell lines. In silico analyses of the FRK promoter region led to the identification of at least 17 CpG sites. Bisulphite sequencing of the promoter region revealed that two of these sites were consistently methylated in FRK-low/negative cell lines and especially in the basal B breast cancer subtype. We further show that treatment of these cells with histone deacetylase inhibitors, Entinostat and Mocetinostat' promoted re-expression of FRK mRNA and protein. Further, using luciferase reporter assays, we show that both GATA3-binding protein FOG1 and constitutively active STAT5A increased the activity of FRK promoter. Together, our results present the first evidence that site-specific promoter methylation contributes to the repression of FRK more so in basal B breast cancers. Our study also highlights the potential clinical significance of targeting FRK using epigenetic drugs specifically in basal B breast cancers which are usually triple negative and very aggressive.

The common R653Q variant (∼20% homozygosity in Caucasians) in the synthetase domain of the folate-metabolizing enzyme MTHFD1 reduces purine synthesis. Although this variant does not appear to affect risk for colorectal cancer, we questioned whether it would affect growth of colorectal tumors. We induced tumor formation in a mouse model for MTHFD1-synthetase deficiency (Mthfd1S

BACKGROUND: Protein tyrosine kinase 6 (PTK6) is overexpressed in many epithelial tumors and predicts poor prognosis. However, PTK6 expression status and its role in cervical squamous cell cancer are unknown. This study aimed to investigate the expression level and clinical significance of PTK6 in early-stage cervical squamous cell cancer.
METHODS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting analysis were performed to detect PTK6 mRNA and protein expression levels in 10 freshly frozen, early-stage cervical squamous cell cancer specimens and adjacent non-tumorous cervical tissues. The expression of PTK6 was detected using immunohistochemical staining in 150 formalin-fixed, paraffin-embedded, early-stage cervical squamous cell cancer sections and 10 normal cervical tissue sections.
RESULTS: The mRNA and protein levels of PTK6 in cancer tissues were higher than those in adjacent non-tumorous cervical tissues. Immunohistochemical analysis showed that PTK6 was not expressed in normal cervical tissues but was overexpressed in the cytoplasm of cervical squamous cell cancer cells. The level of PTK6 expression was significantly associated with tumor grade (P = 0.020). The 5-year overall survival rate of patients with high PTK6 expression was lower than that of patients with low PTK6 expression (81.3% vs. 96.2%, P = 0.008). Multivariate Cox regression analysis showed that the expression level of PTK6 in cervical squamous cell cancer was an independent prognostic factor for patient survival (hazard ratio = 5.999, 95% confidence interval 1.622-22.191, P < 0.05).
CONCLUSIONS: PTK6 is overexpressed in cervical squamous cell cancer. Increased PTK6 expression is associated with reduced 5-year overall survival. PTK6 expression is an independent prognostic predictor for cervical cancer.

BACKGROUND: Cancer has been extensively characterized on the basis of genomics. The integration of genetic information about cancers with data on how the cancers respond to target based therapy to help to optimum cancer treatment.
OBJECTIVE: The increasing usage of sequencing technology in cancer research and clinical practice has enormously advanced our understanding of cancer mechanisms. The cancer precision medicine is becoming a reality. Although off-label drug usage is a common practice in treating cancer, it suffers from the lack of knowledge base for proper cancer drug selections. This eminent need has become even more apparent considering the upcoming genomics data.
METHODS: In this paper, a personalized medicine knowledge base is constructed by integrating various cancer drugs, drug-target database, and knowledge sources for the proper cancer drugs and their target selections. Based on the knowledge base, a bioinformatics approach for cancer drugs selection in precision medicine is developed. It integrates personal molecular profile data, including copy number variation, mutation, and gene expression.
RESULTS: By analyzing the 85 triple negative breast cancer (TNBC) patient data in the Cancer Genome Altar, we have shown that 71.7% of the TNBC patients have FDA approved drug targets, and 51.7% of the patients have more than one drug target. Sixty-five drug targets are identified as TNBC treatment targets and 85 candidate drugs are recommended. Many existing TNBC candidate targets, such as Poly (ADP-Ribose) Polymerase 1 (PARP1), Cell division protein kinase 6 (CDK6), epidermal growth factor receptor, etc., were identified. On the other hand, we found some additional targets that are not yet fully investigated in the TNBC, such as Gamma-Glutamyl Hydrolase (GGH), Thymidylate Synthetase (TYMS), Protein Tyrosine Kinase 6 (PTK6), Topoisomerase (DNA) I, Mitochondrial (TOP1MT), Smoothened, Frizzled Class Receptor (SMO), etc. Our additional analysis of target and drug selection strategy is also fully supported by the drug screening data on TNBC cell lines in the Cancer Cell Line Encyclopedia.
CONCLUSIONS: The proposed bioinformatics approach lays a foundation for cancer precision medicine. It supplies much needed knowledge base for the off-label cancer drug usage in clinics.

The non-receptor tyrosine kinase Fyn-related kinase (FRK) is a member of the BRK family kinases (BFKs) and is distantly related to the Src family kinases (SFKs). FRK was first discovered in 1993, and studies pursued thereafter attributed a potential tumour-suppressive function to the enzyme. In recent years, however, further functional characterization of the tyrosine kinase in diverse cancer types suggests that FRK may potentially play an oncogenic role as well. Specifically, while ectopic expression of FRK suppresses cell proliferation and migration in breast and brain cancers, knockdown or catalytic inhibition of FRK suppresses these cellular processes in pancreatic and liver cancer. Such functional paradox is therefore evidently exhibited in a tissue-specific context. This review sheds light on the recent developments emerged from investigations on FRK which include: (a) a review of the expression pattern of the protein in mammalian cells/tissues, (b) underlying genomic perturbations and (c) a mechanistic function of the enzyme across different cellular environments. Given its functional heterogeneity observed across different cancers, we also discuss the therapeutic significance of FRK.

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.

Cancer cells use stress response pathways to sustain their pathogenic behavior. In breast cancer, stress response-associated phenotypes are mediated by the breast tumor kinase, Brk (PTK6), via the hypoxia-inducible factors HIF-1α and HIF-2α. Given that glucocorticoid receptor (GR) is highly expressed in triple-negative breast cancer (TNBC), we investigated cross-talk between stress hormone-driven GR signaling and HIF-regulated physiologic stress. Primary TNBC tumor explants or cell lines treated with the GR ligand dexamethasone exhibited robust induction of Brk mRNA and protein that was HIF1/2-dependent. HIF and GR coassembled on the BRK promoter in response to either hypoxia or dexamethasone, indicating that Brk is a direct GR/HIF target. Notably, HIF-2α, not HIF-1α, expression was induced by GR signaling, and the important steroid receptor coactivator PELP1 was also found to be induced in a HIF-dependent manner. Mechanistic investigations showed how PELP1 interacted with GR to activate Brk expression and demonstrated that physiologic cell stress, including hypoxia, promoted phosphorylation of GR serine 134, initiating a feed-forward signaling loop that contributed significantly to Brk upregulation. Collectively, our findings linked cellular stress (HIF) and stress hormone (cortisol) signaling in TNBC, identifying the phospho-GR/HIF/PELP1 complex as a potential therapeutic target to limit Brk-driven progression and metastasis in TNBC patients.

Constitutive overactivation of TGFβ signaling is a common event in human cancer progression and acts as a major inducer of epithelial-mesenchymal transition (EMT). In pre-metastatic colorectal cancer (CRC) cells, however, this cascade is tightly controlled and the underlying mechanism in TGFβ stimulated hyperactivation of downstream Smad pathway remains elusive. In this study, expression of miR-187 was downregulated in colorectal cancer (CRC) compared with adjacent normal tissues. miR-187 could suppress the formation of aggressive phenotype in CRC and inactivate Smad pathway, thus preventing EMT. TGFβ stimulation significantly suppressed the expression of miR-187, and overexpressed miR-187 counteracted the influence of TGFβ on cell phenotype and downstream pathway. Furthermore, we found that miR-187 directly suppressed the expression of SOX4, NT5E and PTK6, which were identified as essential upstream effectors of Smad pathway. Together with the fact that high SOX4 or NT5E levels were associated with poor prognosis, we also demonstrated that downregulation of miR-187 was closely related to tumor metastasis and poor prognosis in CRC. These findings revealed a plausible mechanism for sustained TGFβ activation in cancer progression and might have suggested a novel miR-187-based clinical intervention target for patients with advanced CRC.

BACKGROUND: Studies in taxane and/or anthracycline refractory metastatic breast cancer (mBC) patients have shown approximately 30% response rates to irinotecan. Hence, a significant number of patients will experience irinotecan-induced side effects without obtaining any benefit. The aim of this study was to lay the groundwork for development of predictive biomarkers for irinotecan treatment in BC.
METHODS: We established BC cell lines with acquired or de novo resistance to SN-38, by exposing the human BC cell lines MCF-7 and MDA-MB-231 to either stepwise increasing concentrations over 6 months or an initial high dose of SN-38 (the active metabolite of irinotecan), respectively. The resistant cell lines were analyzed for cross-resistance to other anti-cancer drugs, global gene expression, growth rates, TOP1 and TOP2A gene copy numbers and protein expression, and inhibition of the breast cancer resistance protein (ABCG2/BCRP) drug efflux pump.
RESULTS: We found that the resistant cell lines showed 7-100 fold increased resistance to SN-38 but remained sensitive to docetaxel and the non-camptothecin Top1 inhibitor LMP400. The resistant cell lines were characterized by Top1 down-regulation, changed isoelectric points of Top1 and reduced growth rates. The gene and protein expression of ABCG2/BCRP was up-regulated in the resistant sub-lines and functional assays revealed BCRP as a key mediator of SN-38 resistance.
CONCLUSIONS: Based on our preclinical results, we suggest analyzing the predictive value of the BCRP in breast cancer patients scheduled for irinotecan treatment. Moreover, LMP400 should be tested in a clinical setting in breast cancer patients with resistance to irinotecan.

Although long non-coding RNAs (lncRNAs) predominately reside in the nucleus and exert their functions in many biological processes, their potential involvement in cytoplasmic signal transduction remains unexplored. Here, we identify a cytoplasmic lncRNA, LINK-A (long intergenic non-coding RNA for kinase activation), which mediates HB-EGF-triggered, EGFR:GPNMB heterodimer-dependent HIF1α phosphorylation at Tyr 565 and Ser 797 by BRK and LRRK2, respectively. These events cause HIF1α stabilization, HIF1α-p300 interaction, and activation of HIF1α transcriptional programs under normoxic conditions. Mechanistically, LINK-A facilitates the recruitment of BRK to the EGFR:GPNMB complex and BRK kinase activation. The BRK-dependent HIF1α Tyr 565 phosphorylation interferes with Pro 564 hydroxylation, leading to normoxic HIF1α stabilization. Both LINK-A expression and LINK-A-dependent signalling pathway activation correlate with triple-negative breast cancer (TNBC), promoting breast cancer glycolysis reprogramming and tumorigenesis. Our findings illustrate the magnitude and diversity of cytoplasmic lncRNAs in signal transduction and highlight the important roles of lncRNAs in cancer.

Protein tyrosine kinase 6 (PTK6) expression, activation, and amplification of the PTK6 gene have been reported in ERBB2/HER2-positive mammary gland cancers. To explore contributions of PTK6 to mammary gland tumorigenesis promoted by activated ERBB2, we crossed Ptk6-/- mice with the mouse mammary tumor virus-ERBB2 transgenic mouse line expressing activated ERBB2 and characterized tumor development and progression. ERBB2-induced tumorigenesis was significantly delayed and diminished in mice lacking PTK6. PTK6 expression was induced in the mammary glands of ERBB2 transgenic mice before tumor development and correlated with activation of signal transducer and activator of transcription 3 (STAT3) and increased proliferation. Disruption of PTK6 impaired STAT3 activation and proliferation. Phosphorylation of the PTK6 substrates focal adhesion kinase (FAK) and breast cancer anti-estrogen resistance 1 (BCAR1; p130CAS) was decreased in Ptk6-/- mammary gland tumors. Reduced numbers of metastases were detected in the lungs of Ptk6-/- mice expressing activated ERBB2, compared with wild-type ERBB2 transgenic mice. PTK6 activation was detected at the edges of ERBB2-positive tumors. These data support roles for PTK6 in both ERBB2-induced mammary gland tumor initiation and metastasis, and identify STAT3, FAK, and BCAR1 as physiologically relevant PTK6 substrates in breast cancer. Including PTK6 inhibitors as part of a treatment regimen could have distinct benefits in ERBB2/HER2-positive breast cancers.

INTRODUCTION: Protein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase that is highly expressed in Human Epidermal Growth Factor 2(+) (Her2(+)) breast cancers. Overexpression of PTK6 enhances anchorage-independent survival, proliferation, and migration of breast cancer cells. We hypothesized that PTK6 inhibition is an effective strategy to inhibit growth and survival of Her2(+) breast cancer cells, including those that are relatively resistant to Lapatinib, a targeted therapy for Her2(+) breast cancer, either intrinsically or acquired after continuous drug exposure.
METHODS: To determine the effects of PTK6 inhibition on Lapatinib-resistant Her2(+) breast cancer cell lines (UACC893R1 and MDA-MB-453), we used short hairpin ribonucleic acid (shRNA) vectors to downregulate PTK6 expression. We determined the effects of PTK6 downregulation on growth and survival in vitro and in vivo, as well as the mechanisms responsible for these effects.
RESULTS: Lapatinib treatment of "sensitive" Her2(+) cells induces apoptotic cell death and enhances transcript and protein levels of Bim, a pro-apoptotic Bcl2 family member. In contrast, treatment of relatively "resistant" Her2(+) cells fails to induce Bim or enhance levels of cleaved, poly-ADP ribose polymerase (PARP). Downregulation of PTK6 expression in these "resistant" cells enhances Bim expression, resulting in apoptotic cell death. PTK6 downregulation impairs growth of these cells in in vitro 3-D Matrigel(TM) cultures, and also inhibits growth of Her2(+) primary tumor xenografts. Bim expression is critical for apoptosis induced by PTK6 downregulation, as co-expression of Bim shRNA rescued these cells from PTK6 shRNA-induced death. The regulation of Bim by PTK6 is not via changes in Erk/MAPK or Akt signaling, two pathways known to regulate Bim expression. Rather, PTK6 downregulation activates p38, and pharmacological inhibition of p38 activity prevents PTK6 shRNA-induced Bim expression and partially rescues cells from apoptosis.
CONCLUSIONS: PTK6 downregulation induces apoptosis of Lapatinib-resistant Her2(+) breast cancer cells by enhancing Bim expression via p38 activation. As Bim expression is a critical biomarker for response to many targeted therapies, PTK6 inhibition may offer a therapeutic approach to treating patients with Her2 targeted therapy-resistant breast cancers.

UNLABELLED: Protein Tyrosine Kinase 6 (PTK6) is a non-receptor-type tyrosine kinase known to be expressed in various cancers, including pancreatic cancer. The role of PTK6 in cancer chemoresistance remains unclear. Therefore, it was hypothesized that PTK6 mechanistically regulates gemcitabine resistance in pancreatic cancer. Gemcitabine treatment stimulated endogenous PTK6 overexpression in MIAPaCa2 and Panc1 cells. PTK6 gene silencing increased cell survival after gemcitabine treatment and decreased apoptosis, whereas PTK6 overexpression decreased cell survival and increased apoptosis. Selection for gemcitabine resistance revealed substantially lower PTK6 expression in the gemcitabine-resistant subclones compared with the parental lines, while restoring PTK6 rescued gemcitabine sensitivity. Gemcitabine induced phosphorylation of H2AX (γ-H2AX) and ataxia-telangiectasia mutated kinase (pATM), specific markers for DNA double-strand breaks. Both gemcitabine-induced phosphorylation of H2AX and ATM were reduced by PTK6 knockdown and increased by PTK6 overexpression. PTK6 overexpression also increased the S-phase fraction 48 hours after gemcitabine treatment. Although gemcitabine activated both caspase-8 (CASP8) and caspase-9 (CASP9), the effect of PTK6 on gemcitabine-induced apoptosis required CASP8 but not CASP9. In mouse xenografts, PTK6 overexpression in subcutaneous tumors attenuated tumor growth after gemcitabine treatment. In conclusion, PTK6 prolongs S-phase and increases the ability of gemcitabine to cause DNA damage in vitro and in vivo.
IMPLICATIONS: PTK6 affects cell cycle and DNA damage, thus making it an important therapeutic target to improve the outcomes of patients with pancreatic cancer.

Twenty years have passed since the non-receptor tyrosine kinase, Breast tumor kinase (BRK) was cloned. While BRK is evolutionarily related to the Src family kinases it forms its own distinct sub-family referred here to as the BRK family kinases. The detection of BRK in over 60% of breast carcinomas two decades ago and more remarkably, its absence in the normal mammary gland attributed to its recognition as a mammary gland-specific potent oncogene and led BRK researchers on a wild chase to characterize the role of the enzyme in breast cancer. Where has this chase led us? An increasing number of studies have been focused on understanding the cellular roles of BRK in breast carcinoma and normal tissues. A majority of such studies have proposed an oncogenic function of BRK in breast cancers. Thus far, the vast evidence gathered highlights a regulatory role of BRK in critical cellular processes driving tumor formation such as cell proliferation, migration and metastasis. Functional characterization of BRK has identified several signaling proteins that work in concert with the enzyme to sustain such a malignant phenotype. As such targeting the non-receptor tyrosine kinase has been proposed as an attractive approach towards therapeutic intervention. Yet much remains to be explored about (a) the discrepant expression levels of BRK in cancer versus normal conditions, (b) the dependence on the enzymatic activity of BRK to promote oncogenesis and (c) an understanding of the normal physiological roles of the enzyme. This review outlines the advances made towards understanding the cellular and physiological roles of BRK, the mechanisms of action of the protein and its therapeutic significance, in the context of breast cancer.

Brk (breast tumor kinase, also known as PTK6) is a nonreceptor tyrosine kinase that is aberrantly expressed in several cancers and promotes cell proliferation and transformation. Genome sequencing studies have revealed a number of cancer-associated somatic mutations in the Brk gene; however, their effect on Brk activity has not been examined. We analyzed a panel of cancer-associated mutations and determined that several of the mutations activate Brk, while two eliminated enzymatic activity. Three of the mutations (L16F, R131L, and P450L) are located in important regulatory domains of Brk (the SH3, SH2 domains, and C-terminal tail, respectively). Biochemical data suggest that they activate Brk by disrupting intramolecular interactions that normally maintain Brk in an autoinhibited conformation. We also observed differential effects on recognition and phosphorylation of substrates, suggesting that the mutations can influence downstream Brk signaling by multiple mechanisms.

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.

The tyrosine kinome and phosphatome harbor oncogenes and tumor suppressor genes and important regulators of angiogenesis and tumor stroma formation. To provide a better understanding of their potential roles in cancer, we analyzed the expression of 85 tyrosine kinases and 42 tyrosine phosphatases by in situ hybridization 48 human normal and 24 tumor tissue specimens. Nine-tenths of the assessed transcripts had tumor cell expression concordant with expression array databases. Further, pan-cancer expression of AATK, PTPRK, and PTPRU and expression of PTPRS in a subset of tumors were observed. To demonstrate tumor subcompartment resolution, we validated the predicted tumor stroma-specific markers HTRA1, HTRA3, MXRA5, MXRA8, and SERPING1 in situ. In addition to known vascular and stromal markers such as PDGFRB, we observed stromal expression of PTK6 and TNS1 and vascular expression of INSR, PTPRF, PTPRG, PTPRU, and TNS1, of which INSR emerged as a tumor-specific vessel marker. This study demonstrates the feasibility of large-scale analyses to chart the transcriptome in situ in human cancers and their ability to identify novel cancer biomarkers.

Analysis of natural selection events is an attractive strategy for identification of functional variants shaped by gene-environmental interactions and human adaptation. Here, we identified PTK6, a Src-related tyrosine kinase gene, underlying positive selection in East Asian populations. Interestingly, PTK6 variant showed significant correlation with gastric cancer incidences which was the highest in East Asian populations. The high prevalence of gastric cancer in East Asians was also believed to be strongly affected by Helicobacter pylori infection and dietary habit. Therefore, we speculated a competitive interaction of cancer-associated molecules for activation/reduction, where PTK6 likely plays a role through CagA-driven signaling pathway after H. pylori infection. This hypothesis was also supported by our gene expression analysis and the dating of the selective event which was estimated to be ~16,500 years ago, much later than H. pylori invasion in human 50,000 years ago. Establishment of cross talk between PTK6 and CagA by functional studies may further elucidate the underlying biology of H. pylori-mediated gastric cancer.

BACKGROUND & AIMS: Breast tumor kinase (BRK) augments proliferation and promotes cell survival in breast cancers via interactions with SH2 and SH3 ligand-containing proteins, such as receptor tyrosine kinases (RTK; e.g. EGFR, ErbB2/neu). Since RTK contribute to cholangiocarcinoma (CC) evolution we probed BRK protein expression and function in normal and CC livers.
METHODS: Immunohistochemical staining of normal livers and CC (n=93) in a tissue microarray and three CC and an immortalized human cholangiocyte cell lines (real-time PCR, Western blotting, siRNA) were used to study the functional relationships between BRK, EGFR, ErbB2, SAM68, and SPRR2a.
RESULTS: BRK protein was expressed in normal human intrahepatic bile ducts; all CC cell lines and a majority of CC showed strong BRK protein expression. Multiplex immunostaining/tissue cytometry and immunoprecipitation studies showed: 1) BRK co-localized with EGFR and ErbB2/neu; 2) BRK(high)/EGFR(high)-co-expressing CC cells had significantly higher Ki67 labeling and; 3) stronger BRK protein expression was seen in perihilar and distal CC than intrahepatic CC and directly correlated with CC differentiation. In cell lines, BRK expression augmented proliferation in response to exogenous EGF, whereas BRK siRNA significantly reduced growth. The SH3 ligand-containing, SPRR2A activated pTyr342 BRK, which in turn, phosphorylated SAM68, causing nuclear localization and increased cell proliferation similar to observations in breast cancers.
CONCLUSION: BRK expression in a majority of CC can interact with RTK, augmenting growth and interfering with proliferation inhibitors (SAM68). Therapeutically targeting BRK function (in addition to RTK) should be of benefit for CC treatment.

OBJECTIVE: To investigate prostate cancer-related genes and lncRNAs by using a high throughput sequencing dataset.
MATERIALS AND METHODS: RNA-seq data were obtained from the sequencing read archive database, including both benign and malignant tumor samples. After aligning the RNA-seq reads to human genome reference, gene expression profile as well as lncRNA expression profile was obtained. Next, student's t-test was used to screen both the differentially expressed genes (DEGs) and lncRNAs (DELs) between benign and malignant samples. Finally, goseq was used to conduct the functional annotation of DEGs.
RESULTS: A total of 7112 DEGs were screened, such as ZNF512B, UCKL1, STMN3, GMEB2, and PTK6. The top 10 enriched functions of DEGs were mainly related to organism development, including multi-cellular development, system development and anatomical structure development. Also, we discovered 26 differentially expressed lncRNAs.
CONCLUSIONS: The analysis used in this study is reliable in screening prostate cancer markers including both genes and lncRNAs by using RNA-seq data, which provides new insight into the understanding of molecular mechanism of prostate cancer.

To investigate the clinical prognostic value of protein tyrosine kinase 6 (PTK6) in patients with esophageal squamous cell carcinoma (ESCC), quantitative RT-PCR and Western blotting were utilized to measure the mRNA and protein expression levels of PTK6 in 29 and eight pairs of ESCC and peritumoral normal esophageal tissues, respectively. Furthermore, the expression of PTK6 protein in 210 ESCCs was examined with immunohistochemistry (IHC), and its clinical value was analyzed using Kaplan-Meier plots and the Cox proportional hazards regression model. The results found that the expression levels of both PTK6 mRNA and protein in ESCC tissues were significantly lower than those in peritumoral normal esophageal tissues. Regarding the IHC analysis of ESCC, the cytoplasmic expression of PTK6 was significantly correlated with tumor grade (P < 0.001). Compared with patients with low PTK6 expression, ESCC patients with overexpression of PTK6 displayed preferable disease-free survival (DFS) and overall survival (OS) (P < 0.001 and P = 0.001, respectively), especially in stage II disease (P = 0.002 and P = 0.021, respectively). PTK6 was evaluated as an independent prognostic factor for ESCC using multivariate Cox regression analysis. All data demonstrated that the expression level of PTK6 is an independent prognostic factor in ESCCs. Low expression of PTK6 is correlated with poor DFS and OS in ESCCs.

The use of molecularly targeted drugs as single agents has shown limited utility in many tumor types, largely due to the complex and redundant nature of oncogenic signaling networks. Targeting of the PI3K/AKT/mTOR pathway through inhibition of mTOR in combination with aromatase inhibitors has seen success in particular sub-types of breast cancer and there is a need to identify additional synergistic combinations to maximize the clinical potential of mTOR inhibitors. We have used loss-of-function RNAi screens of the mTOR inhibitor rapamycin to identify sensitizers of mTOR inhibition. RNAi screens conducted in combination with rapamycin in multiple breast cancer cell lines identified six genes, AURKB, PLK1, PIK3R1, MAPK12, PRKD2, and PTK6 that when silenced, each enhanced the sensitivity of multiple breast cancer lines to rapamycin. Using selective pharmacological agents we confirmed that inhibition of AURKB or PLK1 synergizes with rapamycin. Compound-associated gene expression data suggested histone deacetylation (HDAC) inhibition as a strategy for reducing the expression of several of the rapamycin-sensitizing genes, and we tested and validated this using the HDAC inhibitor entinostat in vitro and in vivo. Our findings indicate new approaches for enhancing the efficacy of rapamycin including the use of combining its application with HDAC inhibition.

UNLABELLED: Prostate cancer has a proclivity to metastasize to bone. The mechanism by which prostate cancer cells are able to survive and progress in the bone microenvironment is not clear. Identification of molecules that play critical roles in the progression of prostate cancer in bone will provide essential targets for therapy. Ribosomal S6 protein kinases (RSK) have been shown to mediate many cellular functions critical for cancer progression. Whether RSK plays a role in the progression of prostate cancer in bone is unknown. IHC analysis of human prostate cancer specimens showed increased phosphorylation of RSK in the nucleus of prostate cancer cells in a significant fraction of human prostate cancer bone metastasis specimens, compared with the primary site or lymph node metastasis. Expression of constitutively active myristylated RSK in C4-2B4 cells (C4-2B4/RSK) increased their survival and anchorage-independent growth compared with C4-2B4/vector cells. Using an orthotopic bone injection model, it was determined that injecting C4-2B4/RSK cells into mouse femurs enhanced their progression in bone compared with control cells. In PC3-mm2 cells, knockdown of RSK1 (RPS6KA1), the predominant RSK isoform, but not RSK2 (RPS6KA2) alone, decreased anchorage-independent growth in vitro and reduced tumor progression in bone and tumor-induced bone remodeling in vivo. Mechanistic studies showed that RSK regulates anchorage-independent growth through transcriptional regulation of factors that modulate cell survival, including ING3, CKAP2, and PTK6. Together, these data provide strong evidence that RSK is an important driver in prostate cancer progression in bone.
IMPLICATIONS: RSK, an important driver in prostate cancer progression in bone, has promising potential as a therapeutic target for prostate cancer bone metastasis.

Breast and prostate cancers are among the most common cancers worldwide with devastating statistics for the metastatic, chemotherapy- and radiotherapy-resistant phenotypes. Novel therapies interfering with new and/or multiple pathways involved in the pathology of cancer are urgently needed. Preliminary results showed that the marine natural product Z-4-hydroxyphenylmethylene hydantoin (PMH, ) and its 4-ethylthio-analog (SEth, ) promoted tight junction formation and showed anti-invasive and anti-migratory activities in vitro against metastatic prostate cancer cells and inhibited tumor growth and micrometastases in distant organs in orthotopic and transgenic mice models. This study focuses on the design and synthesis of second-generation PMHs with enhanced antitumor activities. A series of substituted benzaldehydes was selected based on earlier SAR studies and reacted with hydantoin to yield 11 new compounds . Compounds were evaluated for their antiproliferative, antimigratory and anti-invasive properties in vitro against the human mammary and prostate cancer cell lines MDA-MB-231 and PC-3, respectively. A Western blot analysis of the most active analog showed its ability to suppress the expression of the total levels of c-Met and FAK, with subsequent reduction of their phosphorylated (activated) levels in MDA-MB-231 cells. In addition, also inhibited Brk, paxillin and Rac1 phosphorylation. was formulated using hydroxypropyl β-cyclodextrin (HPCD) to improve its solubility and was further evaluated in a nude mice xenograft model using MDA-MB-231/GFP cells. PMH reduced breast tumor growth and suppressed Ki-67, CD31, p-Brk and p-FAK expression in tumor samples. Thus, is a potential lead for the control of invasive breast malignancies.

Protein Tyrosine Kinase 6 (PTK6) is a non-receptor type tyrosine kinase that may be involved in some cancers. However, the biological role and expression status of PTK6 in pancreatic cancer is unknown. Therefore in this study, we evaluated the functional role of PTK6 on pancreatic cancer invasion. Five pancreatic cancer cell lines expressed PTK6 at varying levels. PTK6 expression was also observed in human pancreatic adenocarcinomas. PTK6 suppression by siRNA significantly reduced both cellular migration and invasion (0.59/0.49 fold for BxPC3, 0.61/0.62 for Panc1, 0.42/0.39 for MIAPaCa2, respectively, p<0.05 for each). In contrast, forced overexpression of PTK6 by transfection of a PTK6 expression vector in Panc1 and MIAPaCa2 cells increased cellular migration and invasion (1.57/1.67 fold for Panc1, 1.44/1.57 for MIAPaCa2, respectively, p<0.05). Silencing PTK6 reduced ERK1/2 activation, but not AKT or STAT3 activation, while PTK6 overexpression increased ERK1/2 activation. U0126, a specific inhibitor of ERK1/2, completely abolished the effect of PTK6 overexpression on cellular migration and invasion. These results suggest that PTK6 regulates cellular migration and invasion in pancreatic cancer via ERK signaling. PTK6 may be a novel therapeutic target for pancreatic cancer.

Breast tumor kinase (BRK), also known as protein tyrosine kinase 6 (PTK6), is a non-receptor tyrosine kinase overexpressed in more that 60% of human breast carcinomas. The overexpression of BRK has been shown to sensitize mammary epithelial cells to mitogenic signaling and to promote cell proliferation and tumor formation. The molecular mechanisms of BRK have been unveiled by the identification and characterization of BRK target proteins. Downstream of tyrosine kinases 1 or Dok1 is a scaffolding protein and a substrate of several tyrosine kinases. Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362. We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism. Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway.

SRMS (Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) belongs to a family of nonreceptor tyrosine kinases, which also includes breast tumour kinase and Fyn-related kinase. SRMS, similar to breast tumour kinase and Fyn-related kinase, harbours a Src homology 3 and Src homology 2, as well as a protein kinase domain. However, unlike breast tumour kinase and Fyn-related kinase, SRMS lacks a C-terminal regulatory tail but distinctively possesses an extended N-terminal region. Both breast tumour kinase and Fyn-related kinase play opposing roles in cell proliferation and signalling. SRMS, however, is an understudied member of this family. Although cloned in 1994, information on the biochemical, cellular and physiological roles of SRMS remains unreported. The present study is the first to explore the expression pattern of SRMS in breast cancers, its enzymatic activity and autoregulatory elements, and the characterization of docking protein 1 as its first bonafide substrate. We found that, similar to breast tumour kinase, SRMS is highly expressed in most breast cancers compared to normal mammary cell lines and tissues. We generated a series of SRMS point and deletion mutants and assessed enzymatic activity, subcellular localization and substrate recognition. We report for the first time that ectopically-expressed SRMS is constitutively active and that its N-terminal region regulates the enzymatic activity of the protein. Finally, we present evidence indicating that docking protein 1 is a direct substrate of SRMS. Our data demonstrate that, unlike members of the Src family, the enzymatic activity of SRMS is regulated by the intramolecular interactions involving the N-terminus of the enzyme and that docking protein 1 is a bona fide substrate of SRMS.