Verteporfin blocks Clusterin which is required for survival of gastric cancer stem cell by modulating HSP90 function.

Gastric cancer stem cell (GCSC) is implicated in gastric cancer relapse, metastasis and drug resistance. However, the key molecule(s) involved in GCSC survival and the targeting drugs are poorly understood. We discovered increased secreted clusterin (S-Clu) protein expression during the sphere-forming growth of GCSC via mass spectrometry. Overexpression of clusterin was detected in 69/90 (77%) of primary GC tissues and significantly associated with T stage, lymph node metastasis and TNM stage. Depletion of clusterin (Clu, the full-length intracellular clusterin) led to the declustering of GCSC tumorspheres and apoptosis of GCSC. Subsequently, we found clusterin was in complex with heat shock protein 90 beta (HSP90) and involved in regulating the cellular level of HSP90 client proteins. Furthermore, by screening a collection of drugs/inhibitors, we found that verteporfin (VP), a phototherapy drug, blocked clusterin gene expression, decreased the HSP90 client proteins and caused cell death of GCSC. VP treatment is more effective in eradicating GCSCs than in killing GC cells. Both clusterin silencing or VP treatment deterred tumor growth in human GCSC xenografts. These findings collectively suggest that GC patients can promptly benefit from clusterin-targeted therapy as well as VP treatment in combination with or subsequent to conventional chemotherapy for reducing mortality of GC.

PML Recruits TET2 to Regulate DNA Modification and Cell Proliferation in Response to Chemotherapeutic Agent.

Aberrant DNA methylation plays a critical role in the development and progression of cancer. Failure to demethylate and to consequently reactivate methylation-silenced genes in cancer contributes to chemotherapeutic resistance, yet the regulatory mechanisms of DNA demethylation in response to chemotherapeutic agents remain unclear. Here, we show that promyelocytic leukemia (PML) recruits ten-eleven translocation dioxygenase 2 (TET2) to regulate DNA modification and cell proliferation in response to chemotherapeutic agents. TET2 was required by multiple chemotherapeutic agents (such as doxorubicin) to prmote 5-hydroxymethylcytosine (5hmC) formation. Stable isotope labeling with amino acids in cell culture, followed by immunoprecipitation-mass spectrometry, identified potential binding partners of TET2, of which PML mostly enhanced 5hmC formation. PML physically bound to TET2 via the PML C-terminal domain and recruited TET2 to PML-positive nuclear bodies. This interaction was disrupted by the PML-RARA t(15;17) mutation, which stems from chromosomal translocation between DNA encoding the C-terminal domain of PML and the retinoic acid receptor alpha (RARA) gene. In response to chemotherapeutic drugs, PML recruited TET2, regulated DNA modification, reactivated methylation-silenced genes, and impaired cell proliferation. Knockout of PML abolished doxorubicin-promoted DNA modification. In addition, PML and TET2 levels positively correlated with improved overall survival in patients with head and neck cancer. These findings shed insight into the regulatory mechanisms of DNA modification in response to chemotherapeutic agents.Significance: Promyeloctic leukemia protein recruits TET2, regulating DNA modification and cell proliferation in response to chemotherapeutic agents. Cancer Res; 78(10); 2475-89. ©2018 AACR.

Corrigendum: Targeting BMK1 Impairs the Drug Resistance to Combined Inhibition of BRAF and MEK1/2 in Melanoma.

This corrects the article DOI: 10.1038/srep46244.

Targeting BMK1 Impairs the Drug Resistance to Combined Inhibition of BRAF and MEK1/2 in Melanoma.

Combined inhibition of BRAF and MEK1/2 (CIBM) improves therapeutic efficacy of BRAF-mutant melanoma. However, drug resistance to CIBM is inevitable and the drug resistance mechanisms still remain to be elucidated. Here, we show that BMK1 pathway contributes to the drug resistance to CIBM. Considering that ERK1/2 pathway regulates cellular processes by phosphorylating, we first performed a SILAC phosphoproteomic profiling of CIBM. Phosphorylation of 239 proteins was identified to be downregulated, while phosphorylation of 47 proteins was upregulated. Following siRNA screening of 47 upregulated proteins indicated that the knockdown of BMK1 showed the most significant ability to inhibit the proliferation of CIBM resistant cells. It was found that phosphorylation of BMK1 was enhanced in resistant cells, which suggested an association of BMK1 with drug resistance. Further study indicated that phospho-activation of BMK1 by MEK5D enhanced the resistance to CIBM. Conversely, inhibition of BMK1 by shRNAi or BMK1 inhibitor (XMD8-92) impaired not only the acquirement of resistance to CIBM, but also the proliferation of CIBM resistant cells. Further kinome-scale siRNA screening demonstrated that SRC\MEK5 cascade promotes the phospho-activation of BMK1 in response to CIBM. Our study not only provides a global phosphoproteomic view of CIBM in melanoma, but also demonstrates that inhibition of BMK1 has therapeutic potential for the treatment of melanoma.

Inhibition of BMK1 pathway suppresses cancer stem cells through BNIP3 and BNIP3L.

Cancer stem cells (CSCs) possess many characteristics associated with stem cells and are believed to drive tumor initiation. Although targeting of CSCs offers great promise for the new generation of therapeutics, lack of the effective drugable target and appropriate pharmacological reagents significantly impedes the development of chemotherapies. Here, we show that the phosphorylation of BMK1 was significantly correlated with not only embryonic and induced pluripotent stem (iPS) cells, but also the CSCs. It was showed that activation of BMK1 by the expression of MEK5D enhanced the self-renew (sphere formation), proliferation (clone formation) and tumorigenic capacity of CSCs. While BMK1 inhibitor, XMD8-92, suppressed these capacities. RNA-seq and microarray analysis revealed that inhibition of BMK1 significantly enhanced the expression of BNIP3 and BNIP3L, which play important roles in cell death. Further study indicated that shRNA-mediated knock down of BNIP3 and BNIP3L impairs the BMK1 inhibitor, XMD8-92-induced suppression of sphere formation and clone formation of CSC. Collectively, these results not only indicate that BMK1 plays an important role in maintaining "stemness" of CSCs, but also implicate that BMK1 might be a potential drug target for CSCs.

Structural determinants for ERK5 (MAPK7) and leucine rich repeat kinase 2 activities of benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones.

The benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-one core was discovered as a novel ERK5 (also known as MAPK7 and BMK1) inhibitor scaffold, previously. Further structure-activity relationship studies of this scaffold led to the discovery of ERK5-IN-1 (26) as the most selective and potent ERK5 inhibitor reported to date. 26 potently inhibits ERK5 biochemically with an IC50 of 0.162 ± 0.006 µM and in cells with a cellular EC50 for inhibiting epidermal growth factor induced ERK5 autophosphorylation of 0.09 ± 0.03 µM. Furthermore, 26 displays excellent selectivity over other kinases with a KINOMEscan selectivity score (S10) of 0.007, and exhibits exceptional bioavailability (F%) of 90% in mice. 26 will serve as a valuable tool compound to investigate the ERK5 signaling pathway and as a starting point for developing an ERK5 directed therapeutic agent.

Forced expression of miR-143 represses ERK5/c-Myc and p68/p72 signaling in concert with miR-145 in gut tumors of Apc(Min) mice.

Recently, miR-143 and miR-145 have been shown to belong to a subset of microRNAs whose expression is controlled by a complex of a tumor suppressor p53 and DEAD-box RNA helicase subunits p68/p72. While accumulating studies have acknowledged that both miRNAs function as tumor suppressors and are similarly regulated, evidence of their coordinated action against tumorigenesis has been poorly presented. Herein, we establish transgenic mice that express miR-143 under the control of the CAG regulatory unit. When crossbred with Apc(Min/+) mice, the development of tumors in the small intestines is significantly attenuated. In the transgenic small intestine tumors, the endogenous miR-145 is also enhanced and the expression of c-Myc and p68/p72, both of which have been reported to be pivotal for gut tumor development, is suppressed, corresponding to the downregulation of ERK5. We demonstrate that the combination of miR-143 and miR-145 inhibits the expression of c-Myc in human colon cancer cells, whereas miR-145 retards that of p72. Moreover, we show the possibilities that miR-145 modulates p72 expression through its 3' untranslated region and that c-Myc downregulation is involved in both p68 suppression and miR-145 induction. These findings suggest that forced expression of miR-143, probably interacting with endogenous miR-145, inhibits ERK5/c-Myc and p68/p72/ß-catenin signaling and hampers small intestine tumor development in Apc(Min/+) mice. This unique cascade, in turn, may prevent overproduction of a subset of tumor suppressive miRNAs by repressing their own modulators, p68/p72.

BMK1 kinase suppresses epithelial-mesenchymal transition through the Akt/GSK3ß signaling pathway.

Epithelial-mesenchymal transition (EMT) plays a crucial role in the development of cancer metastasis. The mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase, c-jun-NH(2)-kinase, and p38 have been implicated in promoting EMT, but a role for the MAP kinase BMK1 has not been studied. Here, we report that BMK1 signaling suppresses EMT. BMK1 elevation augmented E-cadherin-mediated cell-cell adhesion, downregulated mesenchymal markers, and decreased cell motility. Conversely, BMK1 silencing attenuated E-cadherin-mediated cell-cell adhesion, upregulated mesenchymal markers, and stimulated cell motility. BMK1 depletion dramatically increased the accumulation of endogenous Snail in the nuclear compartment. Snail accumulation was mediated by Akt/GSK3ß signaling, which was activated by a modulation in the expression of the mTOR inhibitor DEPTOR. In support of these observations, BMK1 depletion promoted metastasis in vivo. Together, our findings reveal a novel mechanism of EMT control via mTOR/Akt inhibition that suppresses cancer metastasis.

High-throughput kinase profiling: a more efficient approach toward the discovery of new kinase inhibitors.

Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.

In situ kinase profiling reveals functionally relevant properties of native kinases.

Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.

Targeting the BMK1 MAP kinase pathway in cancer therapy.

The big mitogen activated protein kinase 1 (BMK1) pathway is the most recently discovered and least-studied mammalian mitogen-activated protein (MAP) kinase cascade, ubiquitously expressed in all types of cancer cells tested so far. Mitogens and oncogenic signals strongly activate this cellular MAP kinase pathway, thereby passing down proliferative, survival, chemoresistance, invasive, and angiogenic signals in tumor cells. Recently, several pharmacologic small molecule inhibitors of this pathway have been developed. Among them, the BMK1 inhibitor XMD8-92 blocks cellular BMK1 activation and significantly suppresses tumor growth in lung and cervical tumor models and is well tolerated in animals. On the other hand, MEK5 inhibitors, BIX02188, BIX02189, and compound 6, suppress cellular MEK5 activity, but no data exist to date on their effectiveness in animals.

Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. We employed a new, parallel, compound-centric approach to identify a potent and selective LRRK2 inhibitor, LRRK2-IN-1, and demonstrated that inhibition of LRRK2 induces dephosphorylation of Ser910 and Ser935 and accumulation of LRRK2 within aggregate structures. LRRK2-IN-1 will serve as a versatile tool to pharmacologically interrogate LRRK2 biology and study its role in Parkinson's disease.

Discovery of a benzo[e]pyrimido-[5,4-b][1,4]diazepin-6(11H)-one as a Potent and Selective Inhibitor of Big MAP Kinase 1.

Kinome-wide selectivity profiling of a collection of 2-amino-pyrido[2,3-d]pyrimidines followed by cellular structure-activity relationship-guided optimization resulted in the identification of moderately potent and selective inhibitors of BMK1/ERK5 exemplified by 11, 18, and 21. For example, 11 possesses a dissociation constant (K(d)) for BMK1 of 19 nM, a cellular IC(50) for inhibiting epidermal growth factor induced BMK1 autophosphorylation of 0.19 ± 0.04 µM, and an Ambit KINOMEscan selectivity score (S(5)) of 0.035. Inhibitors 18 and 21 are also potent BMK1 inhibitors and possess favorable pharmacokinetic properties which enable their use as pharmacological probes of BMK1-dependent phenomena as well as starting points for further optimization efforts.

Pharmacological inhibition of BMK1 suppresses tumor growth through promyelocytic leukemia protein.

BMK1 is activated by mitogens and oncogenic signals and, thus, is strongly implicated in tumorigenesis. We found that BMK1 interacted with promyelocytic leukemia protein (PML), and inhibited its tumor-suppressor function through phosphorylation. Furthermore, activated BMK1 notably inhibited PML-dependent activation of p21. To further investigate the BMK-mediated inhibition of the tumor suppressor activity of PML in tumor cells, we developed a small-molecule inhibitor of the kinase activity of BMK1, XMD8-92. Inhibition of BMK1 by XMD8-92 blocked tumor cell proliferation in vitro and significantly inhibited tumor growth in vivo by 95%, demonstrating the efficacy and tolerability of BMK1-targeted cancer treatment in animals.

Kinome siRNA screen identifies SMG-1 as a negative regulator of hypoxia-inducible factor-1alpha in hypoxia.

Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis. To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions. This screen determined that depletion of cellular SMG-1 kinase most significantly modified cellular HIF-1 activity in hypoxia. SMG-1 is the newest and least studied member of the phosphoinositide 3-kinase-related kinase family, which consists of ATM, ATR, DNA-PKcs, mTOR, and SMG-1. We individually depleted members of the phosphoinositide 3-kinase-related kinase family, and only SMG-1 deficiency significantly augmented HIF-1 activity in hypoxia. We subsequently discovered that SMG-1 kinase activity was activated by hypoxia, and depletion of SMG-1 up-regulated MAPK activity under low oxygen. Suppressing cellular MAPK by silencing ERK1/2 or by treatment with U0126, a MAPK inhibitor, partially blocked the escalation of HIF-1 activity resulting from SMG-1 deficiency in hypoxic cells. Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1alpha. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1alpha-regulated angiogenic factor, vascular epidermal growth factor, and survival factor, carbonic anhydrase IX (CA9), as well as promoted the hypoxic cell motility. Taken together, we discovered that SMG-1 negatively regulated HIF-1alpha activity in hypoxia, in part through blocking MAPK activation.

Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration.

Integrins interact with extracellular matrix (ECM) and deliver intracellular signaling for cell proliferation, survival, and motility. During tumor metastasis, integrin-mediated cell adhesion to and migration on the ECM proteins are required for cancer cell survival and adaptation to the new microenvironment. Using stable isotope labeling by amino acids in cell culture-mass spectrometry, we profiled the phosphoproteomic changes induced by the interactions of cell integrins with type I collagen, the most common ECM substratum. Integrin-ECM interactions modulate phosphorylation of 517 serine, threonine, or tyrosine residues in 513 peptides, corresponding to 357 proteins. Among these proteins, 33 key signaling mediators with kinase or phosphatase activity were subjected to small interfering RNA-based functional screening. Three integrin-regulated kinases, DBF4, PAK2, and GRK6, were identified for their critical role in cell adhesion and migration possibly through their regulation of actin cytoskeleton arrangement. Altogether, we not only depict an integrin-modulated phosphorylation network during cell-ECM protein interactions but also reveal novel regulators for cell adhesion and migration.

CDC25B mediates rapamycin-induced oncogenic responses in cancer cells.

Because the mammalian target of rapamycin (mTOR) pathway is commonly deregulated in human cancer, mTOR inhibitors, rapamycin and its derivatives, are being actively tested in cancer clinical trials. Clinical updates indicate that the anticancer effect of these drugs is limited, perhaps due to rapamycin-dependent induction of oncogenic cascades by an as yet unclear mechanism. As such, we investigated rapamycin-dependent phosphoproteomics and discovered that 250 phosphosites in 161 cellular proteins were sensitive to rapamycin. Among these, rapamycin regulated four kinases and four phosphatases. A siRNA-dependent screen of these proteins showed that AKT induction by rapamycin was attenuated by depleting cellular CDC25B phosphatase. Rapamycin induces the phosphorylation of CDC25B at Serine375, and mutating this site to Alanine substantially reduced CDC25B phosphatase activity. Additionally, expression of CDC25B (S375A) inhibited the AKT activation by rapamycin, indicating that phosphorylation of CDC25B is critical for CDC25B activity and its ability to transduce rapamycin-induced oncogenic AKT activity. Importantly, we also found that CDC25B depletion in various cancer cell lines enhanced the anticancer effect of rapamycin. Together, using rapamycin phosphoproteomics, we not only advance the global mechanistic understanding of the action of rapamycin but also show that CDC25B may serve as a drug target for improving mTOR-targeted cancer therapies.

Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis.

Immobilized metal affinity chromatography (IMAC) is a common strategy used for the enrichment of phosphopeptides from digested protein mixtures. However, this strategy by itself is inefficient when analyzing complex protein mixtures. Here, we assess the effectiveness of using protein-based IMAC as a pre-enrichment step prior to peptide-based IMAC. Ultimately, we couple the two IMAC-based enrichments and MudPIT in a quantitative phosphoproteomic analysis of the epidermal growth factor pathway in mammalian cells identifying 4470 unique phosphopeptides containing 4729 phosphorylation sites.

Protein kinase-mediated regulation of calcineurin through the phosphorylation of modulatory calcineurin-interacting protein 1.

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes such as T-cell activation, skeletal myocyte differentiation, and cardiac hypertrophy. We previously showed that active MEKK3 is capable of stimulating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes through phosphorylation of modulatory calcineurin-interacting protein 1 (MCIP1). However, the protein kinases that function downstream of MEKK3 to mediate MCIP1 phosphorylation and the mechanism of MCIP1-mediated calcineurin regulation have not been defined. Here, we show that MEK5 and big MAP kinase 1 (BMK1) function downstream of MEKK3 in a signaling cascade that induces calcineurin activity through phosphorylation of MCIP1. Genetic studies showed that BMK1-deficient mouse lung fibroblasts failed to mediate MCIP1 phosphorylation and activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring BMK1 to the deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. Thus, using BMK1-deficient mouse lung fibroblast cells, we provided the genetic evidence that BMK1 is required for angiotensin II-mediated calcineurin/NFAT activation through MICP1 phosphorylation. Finally, we discovered that phosphorylated MCIP1 dissociates from calcineurin and binds with 14-3-3, thereby relieving its inhibitory effect on calcineurin activity. In summary, our findings reveal a previously unrecognized essential regulatory role of mitogen-activated protein kinase signaling in calcineurin activation through the reversible phosphorylation of a calcineurin-interacting protein, MCIP1.

A crucial role of mitochondrial Hsp40 in preventing dilated cardiomyopathy.

Many heat-shock proteins (Hsp) are members of evolutionarily conserved families of chaperone proteins that inhibit the aggregation of unfolded polypeptides and refold denatured proteins, thereby remedying phenotypic effects that may result from protein aggregation or protein instability. Here we report that the mitochondrial chaperone Hsp40, also known as Dnaja3 or Tid1, is differentially expressed during cardiac development and pathological hypertrophy. Mice deficient in Dnaja3 developed dilated cardiomyopathy (DCM) and died before 10 weeks of age. Progressive respiratory chain deficiency and decreased copy number of mitochondrial DNA were evident in cardiomyocytes lacking Dnaja3. Profiling of Dnaja3-interacting proteins identified the alpha-subunit of DNA polymerase gamma (Polga) as a client protein. These findings suggest that Dnaja3 is crucial for mitochondrial biogenesis, at least in part, through its chaperone activity on Polga and provide genetic evidence of the necessity for mitochondrial Hsp40 in preventing DCM.