Bojie Dai

The 44 kDa Pim-1 kinase directly interacts with tyrosine kinase Etk/BMX and protects human prostate cancer cells from apoptosis induced by chemotherapeutic drugs.

Protein kinase Pim-1 has been implicated in the development of hematopoietic and prostatic malignancies. Here, we present the evidence that two isoforms, the 44 and 33 kDa Pim-1, are expressed in all human prostate cancer cell lines examined. The subcellular localization of human 44 kDa Pim-1 is primarily on the plasma membrane, while the 33 kDa isoform is present in both the cytosol and nucleus in PCA cells. The 44 kDa Pim-1 contains the proline-rich motif at the N-terminus and directly binds to the SH3 domain of tyrosine kinase Etk. Such interaction leads to the activation of Etk kinase activity possibly by competing with the tumor suppressor p53. This is corroborated by the fact that overexpression of the 44 kDa Pim-1 in prostate cancer cells confers the resistance to chemotherapeutic drugs. Our results suggest that these two isoforms of Pim-1 kinase may regulate distinct substrates and the 44 kDa Pim-1 may play a more prominent role in drug resistance in prostate cancer cells.

Compensatory Upregulation of Tyrosine Kinase Etk/BMX in Response to Androgen Deprivation Promotes Castration-Resistant Growth of Prostate Cancer Cells

We previously showed that targeted expression of non-receptor tyrosine kinase Etk/BMX in mouse prostate induces prostate intraepithelial neoplasia, implying a possible causal role of Etk in prostate cancer development and progression. Here, we report that Etk is upregulated in both human and mouse prostates in response to androgen ablation. Etk expression seems to be differentially regulated by androgen and interleukin 6 (IL-6), which is possibly mediated by the androgen receptor (AR) in prostate cancer cells. Our immunohistochemical analysis of tissue microarrays containing 112 human prostate tumor samples revealed that Etk expression is elevated in hormone-resistant prostate cancer and positively correlated with tyrosine phosphorylation of AR (Pearson correlation coefficient rho = 0.71, P < 0.0001). AR tyrosine phosphorylation is increased in Etk-overexpressing cells, suggesting that Etk may be another tyrosine kinase, in addition to Src and Ack-1, which can phosphorylate AR. We also showed that Etk can directly interact with AR through its Src homology 2 domain, and such interaction may prevent the association of AR with Mdm2, leading to stabilization of AR under androgen-depleted conditions. Overexpression of Etk in androgen-sensitive LNCaP cells promotes tumor growth while knocking down Etk expression in hormone-insensitive prostate cancer cells by a specific shRNA that inhibits tumor growth under androgen-depleted conditions. Taken together, our data suggest that Etk may be a component of the adaptive compensatory mechanism activated by androgen ablation in prostate and may play a role in hormone resistance, at least in part, through direct modulation of the AR signaling pathway.

Tyrosine kinase Etk/BMX is up-regulated in human prostate cancer and its overexpression induces prostate intraepithelial neoplasia in mouse

The nonreceptor tyrosine kinase Etk/BMX was originally identified from the human prostate xenograft CWR22. Here, we report that Etk is up-regulated in human prostate tumor specimens surveyed. Knocking down Etk expression by a specific small interfering RNA (siRNA) in prostate cancer cells attenuates cell proliferation, suggesting an essential role of Etk for prostate cancer cell survival and growth. Targeted expression of Etk in mouse prostate epithelium results in pathologic changes resembling human prostatic intraepithelial neoplasia, indicating that up-regulation of Etk may contribute to prostate cancer development. A marked increase of luminal epithelial cell proliferation was observed in the Etk transgenic prostate, which may be attributed in part to the elevated activity of Akt and signal transducers and activators of transcription 3 (STAT3). More interestingly, the expression level of acetyltransferase cyclic AMP-responsive element binding protein-binding protein (CBP) is also increased in the Etk transgenic prostate as well as in a prostate cancer cell line overexpressing Etk, concomitant with elevated histone 3 acetylation at lysine 18 (H3K18Ac). Down-modulation of Etk expression by a specific siRNA leads to a decrease of H3 acetylation in prostate cancer cell lines. Our data suggest that Etk may also modulate chromatin remodeling by regulating the activity of acetyltransferases, such as CBP. Given that Etk may exert its effects in prostate through modulation of multiple signaling pathways altered in human prostate cancer, the Etk transgenic mouse model may be a useful tool for studying the functions of Etk and identification of new molecular markers and drug targets relevant to human diseases.

Bi-directional regulation between tyrosine kinase Etk/BMX and tumor suppressor p53 in response to DNA damage.

Etk/Bmx, a member of the Tec family of nonreceptor tyrosine kinases, has been implicated in the regulation of various cellular processes including proliferation, differentiation, motility, and apoptosis. Here, we report the identification of Tec family kinases as the potential interacting proteins of the tumor suppressor p53 by an Src homology 3 domain array screening. Etk is physically associated with p53 through its Src homology 3 domain and the proline-rich domain of p53. Induction of p53 expression by DNA damage inhibits Etk activity in several cell types. Down-regulation of Etk expression by a specific small interfering RNA sensitizes prostate cancer cells to doxorubicin-induced apoptosis, suggesting that inhibition of Etk activity is required for apoptosis in response to DNA damage. We also show that Etk primarily interacts with p53 in the cytoplasm and that such interaction leads to bidirectional inhibition of the activities of both proteins. Overexpression of Etk in prostate cancer cells results in inhibition of p53 transcriptional activity and its interaction with the mitochondrial protein BAK and confers the resistance to doxorubicin. Therefore, we propose that the stoichiometry between p53 and the Tec family kinases in a given cell type may determine its sensitivity to chemotherapeutic drugs.

ATM regulates a DNA damage response posttranscriptional RNA operon in lymphocytes

Maintenance of genomic stability depends on the DNA damage response, a biologic barrier in early stages of cancer development. Failure of this response results in genomic instability and high predisposition toward lymphoma, as seen in patients with ataxia-telangiectasia mutated (ATM) dysfunction. ATM activates multiple cell-cycle checkpoints and DNA repair after DNA damage, but its influence on posttranscriptional gene expression has not been examined on a global level. We show that ionizing radiation modulates the dynamic association of the RNA-binding protein HuR with target mRNAs in an ATM-dependent manner, potentially coordinating the genotoxic response as an RNA operon. Pharmacologic ATM inhibition and use of ATM-null cells revealed a critical role for ATM in this process. Numerous mRNAs encoding cancer-related proteins were differentially associated with HuR depending on the functional state of ATM, in turn affecting expression of encoded proteins. The findings presented here reveal a previously unidentified role of ATM in controlling gene expression posttranscriptionally. Dysregulation of this DNA damage response RNA operon is probably relevant to lymphoma development in ataxia-telangiectasia persons. These novel RNA regulatory modules and genetic networks provide critical insight into the function of ATM in oncogenesis.

Identification of Transformation-Related Pathways in a Breast Epithelial Cell Model Using a Ribonomics Approach

The aberrant expression of many genes is a common feature in the malignant transformation of cells. In mammalian cells, posttranscriptional gene regulatory processes are emerging as critical determinants controlling gene expression both in physiologic and pathologic conditions. These regulatory mechanisms are directed primarily by the interaction of mRNAs with specific RNA-binding proteins (RBP). There is an emerging body of data demonstrating that two RBPs, AUF1 and HuR, can antagonistically affect the posttranscriptional fate of target mRNAs, as well as concurrently bind to common target transcripts. Employing MCT-1 oncogene-mediated transformation of immortalized breast epithelial MCF10A cells, we characterized the largely reciprocal association of these two RBPs with target mRNAs and their influence on protein expression vis-a-vis cellular transformation. Using a ribonomics approach, we identified mRNAs from cancer-related pathways whose association with AUF1 and/or HuR were altered when comparing immortalized with transformed MCF10A cells. Significantly, we were able to show that knockdown of HuR expression using RNA interference reduced anchorage-independent growth capacity in transformed MCF10A cells and decreased protein expression of a number of validated target genes. Our data show that the global alterations in binding of HuR and AUF1 with target transcripts have a critical role in posttranscriptional regulation of genes encoding proteins involved in breast epithelial cell transformation. These findings further support the feasibility of using a ribonomics approach for the identification of cancer-related pathways.

Functional and molecular interactions between ERK and CHK2 in diffuse large B-cell lymphoma.

Distinct oncogenic signalling cascades have been associated with non-Hodgkin lymphoma. ERK1/2 signalling elicits both transcriptional and post-transcriptional effects through phosphorylation of numerous substrates. Here we report a novel molecular relationship between ERK1/2 and CHK2, a protein kinase that is a key mediator of the DNA damage checkpoint that responds to DNA double-strand breaks. Our studies are the first to demonstrate the co-localization and overexpression of ERK1/2 and CHK2 in diffuse large B-cell lymphoma (DLBCL). The physical interaction between ERK and CHK2 was highly dependent on phosphorylated Thr 68 of CHK2. Concurrent administration of an ERK inhibitor enhances the antitumour activity of CHK2 inhibition in both a human DLBCL xenograft model as well as primary human DLBCL cells. Our data suggest a functional interaction between ERK and CHK2 and support the potential combined therapeutic targeting of ERK and CHK2 in human DLBCL.

MNKs act as a regulatory switch for eIF4E1 and eIF4E3 driven mRNA translation in DLBCL

The phosphorylation of eIF4E1 at serine 209 by MNK1 or MNK2 has been shown to initiate oncogenic mRNA translation, a process that favours cancer development and maintenance. Here, we interrogate the MNK-eIF4E axis in diffuse large B-cell lymphoma (DLBCL) and show a distinct distribution of MNK1 and MNK2 in germinal centre B-cell (GCB) and activated B-cell (ABC) DLBCL. Despite displaying a differential distribution in GCB and ABC, both MNKs functionally complement each other to sustain cell survival. MNK inhibition ablates eIF4E1 phosphorylation and concurrently enhances eIF4E3 expression. Loss of MNK protein itself downregulates total eIF4E1 protein level by reducing eIF4E1 mRNA polysomal loading without affecting total mRNA level or stability. Enhanced eIF4E3 expression marginally suppresses eIF4E1-driven translation but exhibits a unique translatome that unveils a novel role for eIF4E3 in translation initiation. We propose that MNKs can modulate oncogenic translation by regulating eIF4E1-eIF4E3 levels and activity in DLBCL.

Ribosomal protein S6 is highly expressed in non-Hodgkin lymphoma and associates with mRNA containing a 5′ terminal oligopyrimidine tract

The molecular mechanism(s) linking tumorigenesis and morphological alterations in the nucleolus are presently coming into focus. The nucleolus is the cellular organelle in which the formation of ribosomal subunits occurs. Ribosomal biogenesis occurs through the transcription of ribosomal RNA (rRNA), rRNA processing and production of ribosomal proteins. An error in any of these processes may lead to deregulated cellular translation, evident in multiple cancers and ‘ribosomopathies’. Deregulated protein synthesis may be achieved through the overexpression of ribosomal proteins as seen in primary leukemic blasts with elevated levels of ribosomal proteins S11 and S14. In this study, we demonstrate that ribosomal protein S6 (RPS6) is highly expressed in primary diffuse large B-cell lymphoma (DLBCL) samples. Genetic modulation of RPS6 protein levels with specifically targeted short hairpin RNA (shRNA) lentiviruses led to a decrease in the actively proliferating population of cells compared with control shRNA. Low-dose rapamycin treatments have been shown to affect the translation of 5′ terminal oligopyrimidine (5′ TOP) tract mRNA, which encodes the translational machinery, implicating RPS6 in 5′ TOP translation. Recently, it was shown that disruption of 40S ribosomal biogenesis through specific small inhibitory RNA knockdown of RPS6 defined RPS6 as a critical regulator of 5′ TOP translation. For the first time, we show that RPS6 associates with multiple mRNAs containing a 5′ TOP tract. These findings expand our understanding of the mechanism(s) involved in ribosomal biogenesis and deregulated protein synthesis in DLBCL.

The novel anti-MEK small molecule AZD6244 induces BIM-dependent and AKT-independent apoptosis in diffuse large B-cell lymphoma

The RAS/RAF/MEK/ERK signaling pathway has been largely unexplored as a potential therapeutic target in lymphoma. The novel 2nd generation anti-MEK small molecule, AZD6244, down-regulated its direct downstream target, phospho-ERK (pERK) in germinal center and nongerminal center diffuse large B-cell lymphoma (DLBCL) cell lines and primary cells. Similar decreased pERK levels were noted despite constitutive activation (CA) of MEK. Consequently, several lymphoma-related ERK substrates were down-regulated by AZD6244 including MCT-1, c-Myc, Bcl-2, Mcl-1, and CDK1/2. AZD6244 induced time- and dose-dependent antiproliferation and apoptosis in all DLBCL cell lines and fresh/primary cells (IC(50) 100nM-300nM). Furthermore, AZD6244 resulted in significantly less tumor compared with control in an in vivo DLBCL SCID xenograft model. Cell death was associated with cleaved PARP, caspases-8, -9, and -3, and apoptosis was caspase-dependent. In addition, there was stabilization of FoxO3a, activation of BIM and PUMA, and a significant decrease in c-Myc transcripts. Moreover, siRNA knockdown of BIM abrogated AZD6244-related apoptosis, while shRNA knockdown of ERK minimally sensitized cells. Finally, manipulation of AKT with transfection of OCI-LY3 cells with CA-AKT or through chemical inhibition (LY294002) had minimal effect on AZD6244-induced cell death. Altogether, these findings show that the novel anti-MEK agent, AZD6244, induced apoptosis in DLBCL and that cell death was BIM-dependent.