A phosphoarray platform is capable of personalizing kinase inhibitor therapy in head and neck cancers.

Tyrosine kinase inhibitors are effective treatments for cancers. Knowing the specific kinase mutants that drive the underlying cancers predict therapeutic response to these inhibitors. Thus, the current protocol for personalized cancer therapy involves genotyping tumors in search of various driver mutations and subsequently individualizing the tyrosine kinase inhibitor to the patients whose tumors express the corresponding driver mutant. While this approach works when known driver mutations are found, its limitation is the dependence on driver mutations as predictors for response. To complement the genotype approach, we hypothesize that a phosphoarray platform is equally capable of personalizing kinase inhibitor therapy. We selected head and neck squamous cell carcinoma as the cancer model to test our hypothesis. Using the receptor tyrosine kinase phosphoarray, we identified the phosphorylation profiles of 49 different tyrosine kinase receptors in five different head and neck cancer cell lines. Based on these results, we tested the cell line response to the corresponding kinase inhibitor therapy. We found that this phosphoarray accurately informed the kinase inhibitor response profile of the cell lines. Next, we determined the phosphorylation profiles of 39 head and neck cancer patient derived xenografts. We found that absent phosphorylated EGFR signal predicted primary resistance to cetuximab treatment in the xenografts without phosphorylated ErbB2. Meanwhile, absent ErbB2 signaling in the xenografts with phosphorylated EGFR is associated with a higher likelihood of response to cetuximab. In summary, the phosphoarray technology has the potential to become a new diagnostic platform for personalized cancer therapy.

Analysis of the tumor-initiating and metastatic capacity of PDX1-positive cells from the adult pancreas.

Pancreatic cancer is one of the deadliest human malignancies. A striking feature of pancreatic cancer is that activating Kras mutations are found in ∼90% of cases. However, apart from a restricted population of cells expressing pancreatic and duodenal homeobox 1 (PDX1), most pancreatic cells are refractory to Kras-driven transformation. In the present study, we sought to determine which subsets of PDX1+ cells may be responsible for tumor growth. Using the Lox-Stop-Lox-KrasG12D genetic mouse model of pancreatic carcinogenesis, we isolated a population of KrasG12D-expressing PDX1+ cells with an inherent capacity to metastasize. This population of cells bears the surface phenotype of EpCAM+CD24+CD44+CD133-SCA1- and is closer in its properties to stem-like cells than to more mature cell types. We further demonstrate that the tumorigenic capacity of PDX1+ cells is limited, becoming progressively lost as the cells acquire a mature phenotype. These data are consistent with the hypothesis that the adult pancreas harbors a dormant progenitor cell population that is capable of initiating tumor growth under conditions of oncogenic stimulation. We present evidence that constitutive activation of the mitogen-activated protein kinase (MAPK/ERK) signaling and stabilization of the MYC protein are the two main driving forces behind the development of pancreatic cancer cells with stem-cell-like properties and high metastatic potential. Our results suggest that pancreatic cells bearing Kras mutation can be induced to differentiate into quasi-normal cells with suppressed tumorigenicity by selective inhibition of the MAPK/ERK/MYC signaling cascade.

The Ski Protein is Involved in the Transformation Pathway of Aurora Kinase A.

Oncogenic kinase Aurora A (AURKA) has been found to be overexpresed in several tumors including colorectal, breast, and hematological cancers. Overexpression of AURKA induces centrosome amplification and aneuploidy and it is related with cancer progression and poor prognosis. Here we show that AURKA phosphorylates in vitro the transcripcional co-repressor Ski on aminoacids Ser326 and Ser383. Phosphorylations on these aminoacids decreased Ski protein half-life. Reduced levels of Ski resulted in centrosomes amplification and multipolar spindles formation, same as AURKA overexpressing cells. Importantly, overexpression of Ski wild type, but not S326D and S383D mutants inhibited centrosome amplification and cellular transformation induced by AURKA. Altogether, these results suggest that the Ski protein is a target in the transformation pathway mediated by the AURKA oncogene.

Ski-interacting protein (SKIP) interacts with androgen receptor in the nucleus and modulates androgen-dependent transcription.

BACKGROUND: The androgen receptor (AR) is a member of the nuclear receptor (NR) superfamily of ligand-inducible DNA transcription factors, and is the major mediator of male sexual development, prostate growth and the pathogenesis of prostate cancer. Cell and gene specific regulation by the AR is determined by availability of and interaction with sets of key accessory cofactors. Ski-interacting protein (SKIP; SNW1, NCOA62) is a cofactor shown to interact with several NRs and a diverse range of other transcription factors. Interestingly, SKIP as part of the spliceosome is thought to link mRNA splicing with transcription. SKIP has not been previously shown to interact with the AR. RESULTS: The aim of this study was to investigate whether SKIP interacts with the AR and modulates AR-dependent transcription. Here, we show by co-immunoprecipitation experiments that SKIP is in a complex with the AR. Moreover, SKIP increased 5α-dihydrotestosterone (DHT) induced N-terminal/C-terminal AR interaction from 12-fold to almost 300-fold in a two-hybrid assay, and enhanced AR ligand-independent AF-1 transactivation. SKIP augmented ligand- and AR-dependent transactivation in PC3 prostate cancer cells. Live-cell imaging revealed a fast (half-time=129 s) translocation of AR from the cytoplasm to the nucleus upon DHT-stimulation. Förster resonance energy transfer (FRET) experiments suggest a direct AR-SKIP interaction in the nucleus upon translocation. CONCLUSIONS: Our results suggest that SKIP interacts with AR in the nucleus and enhances AR-dependent transactivation and N/C-interaction supporting a role for SKIP as an AR co-factor.

Chromosomal instability in mouse embryonic fibroblasts null for the transcriptional co-repressor Ski.

Ski is a transcriptional regulator that has been considered an oncoprotein given its ability to induce oncogenic transformation in avian model systems. However, studies in mouse and in some human tumor cells have also indicated a tumor suppressor activity for this protein. We found that Ski-/- mouse embryo fibroblasts exhibit high levels of genome instability, namely aneuploidy, consistent with a tumor suppressor function for Ski. Time-lapse microscopy revealed lagging chromosomes and chromatin/chromosome bridges as the major cause of micronuclei (MN) formation and the subsequent aneuploidy. Although these cells arrested in mitosis after treatment with spindle disrupting drugs and exhibited a delayed metaphase/anaphase transition, spindle assembly checkpoint (SAC) was not sufficient to prevent chromosome missegregation, consistent with a weakened SAC. Our in vivo analysis also showed dynamic metaphase plate rearrangements with switches in polarity in cells arrested in metaphase. Importantly, after ectopic expression of Ski the cells that displayed this metaphase arrest died directly during metaphase or after aberrant cell division, relating SAC activation and mitotic cell death. This increased susceptibility to undergo mitosis-associated cell death reduced the number of MN-containing cells. The presented data support a new role for Ski in the mitotic process and in maintenance of genetic stability, providing insights into the mechanism of tumor suppression mediated by this protein.

Identification of Ski as a target for Aurora A kinase.

Ski is a negative regulator of the transforming growth factor-ß and other signalling pathways. The absence of SKI in mouse fibroblasts leads to chromosome segregation defects and genomic instability, suggesting a role for Ski during mitosis. At this stage, Ski is phosphorylated but to date little is known about the kinases involved in this process. Here, we show that Aurora A kinase is able to phosphorylate Ski in vitro. In vivo, Aurora A and Ski co-localized at the centrosomes and co-immunoprecipitated. Conversely, a C-terminal truncation mutant of Ski (SkiΔ491-728) lacking a coiled-coil domain, displayed decreased centrosomal localization. This mutant no longer co-immunoprecipitated with Aurora-A in vivo, but was still phosphorylated in vitro, indicating that the Ski-Aurora A interaction takes place at the centrosomes. These data identify Ski as a novel target of Aurora A and contribute to an understanding of the role of these proteins in the mitotic process.

KRAS drives immune evasion in a genetic model of pancreatic cancer.

Immune evasion is a hallmark of KRAS-driven cancers, but the underlying causes remain unresolved. Here, we use a mouse model of pancreatic ductal adenocarcinoma to inactivate KRAS by CRISPR-mediated genome editing. We demonstrate that at an advanced tumor stage, dependence on KRAS for tumor growth is reduced and is manifested in the suppression of antitumor immunity. KRAS-deficient cells retain the ability to form tumors in immunodeficient mice. However, they fail to evade the host immune system in syngeneic wild-type mice, triggering strong antitumor response. We uncover changes both in tumor cells and host immune cells attributable to oncogenic KRAS expression. We identify BRAF and MYC as key mediators of KRAS-driven tumor immune suppression and show that loss of BRAF effectively blocks tumor growth in mice. Applying our results to human PDAC we show that lowering KRAS activity is likewise associated with a more vigorous immune environment.

The Ski protein negatively regulates Siah2-mediated HDAC3 degradation.

Ski acts as a transcriptional co-repressor by multiple direct and indirect interactions with several distinct repression complexes. Ski represses retinoic acid (RA) signaling by interacting with, and stabilizing, key components of the co-repressor complex, namely, HDAC3. However, little is known as to how the Ski protein can stabilize HDAC3. In the present study, we identified the Siah2 protein as a potential E3 ubiquitin ligase that mediated proteasomal degradation of HDAC3. Reciprocal co-immunoprecipitation assays further revealed that Ski interacts with Siah2. Furthermore, co-expression of the Ski protein stabilized the level of Siah2 protein. Since Siah2 regulates its own level of expression by self-degradation, the stabilization of Siah2 by Ski is an indication that Ski association leads to inhibition of Siah2 E3 ubiquitin ligase activity. Only wild-type Ski and Ski truncation mutants that were in the same complex with Siah2 could stabilize HDAC3 levels. Taken together, the results suggest that association with Ski leads to inhibition of Siah2 E3 ubiquitin ligase activity and in this way, the Ski protein inhibits Siah2-mediated proteasomal degradation of HDAC3.

RON-expressing MCF-10A breast epithelial cells exhibit alterations of hyaluronan expression, promoting RON-mediated early adhesion events.

The receptor tyrosine kinase known as RON appears to play a role in the progression of human carcinomas, and is associated with a poor patient prognosis. Our current study demonstrates that RON expression in MCF-10A breast epithelial cells lead to an alteration of cell-surface hyaluronan compared to the parental cells. We found that hyaluronan was important for initial cell attachment to poly-d-lysine-coated coverslips, but did not contribute to the process of cell spreading. Previous data implied that the Src kinase was important for spreading but not the initial attachment of 10A cells, and here we demonstrate Src activation was also not necessary for hyaluronan production in these cells.

Combination of phosphorylated and truncated EGFR correlates with higher tumor and nodal stage in head and neck cancer.

Epidermal growth factor receptor (EGFR) is a target in head and neck cancer. High EGFR expression and phosphorylated EGFR predicts poor survival in head and neck cancer patients, but does not correlate with advanced stage disease. The aim of this study is to determine if clinical biological correlates are more accurate when different aspects of EGFR are evaluated in combination. We analyzed the EGFR phosphorylation, expression, and mutations in 60 primary head and neck tumors. We not only found that head and neck tumors with either truncated or activated EGFR tend to have higher tumor and nodal stage but also discovered two novel EGFR truncations.

The Ski protein can inhibit ligand induced RARalpha and HDAC3 degradation in the retinoic acid signaling pathway.

Recent data has implicated the Ski protein as being a physiologically relevant negative regulator of signaling by retinoic acid (RA). The mechanism by which Ski represses RA signaling is unknown. Co-immunoprecipitation and immunofluorescence assay showed that Ski and RARalpha are in the same complex in both the absence and presence of RA, which makes Ski different from other corepressors. We determined that Ski can stabilize RARalpha and HDAC3. These results suggest that Ski represses RA signaling by stabilizing corepressor complex.

Therapeutic implications of a human neutralizing antibody to the macrophage-stimulating protein receptor tyrosine kinase (RON), a c-MET family member.

RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in approximately 100 cancer cell lines and approximately 300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers.

Mutation of Thr466 in SHP2 abolishes its phosphatase activity, but provides a new substrate-trapping mutant.

Most classical phosphotyrosyl phosphatases (PTPs), including the Src homology phosphotyrosyl phosphatase 2 (SHP2) possess a Thr or a Ser residue immediately C-terminal to the invariant Arg in the active site consensus motif (H/V-C-X5-R-S/T), also known as the "signature motif". SHP2 has a Thr (Thr466) at this position, but its importance in catalysis has not been investigated. By employing site-directed mutagenesis, phosphatase assays and substrate-trapping studies, we demonstrate that Thr466 is critical for the catalytic activity of SHP2. Its mutation to Ala abolishes phosphatase activity, but provides a new substrate-trapping mutant. We further show that the nucleophilic Cys459 is not involved in substrate trapping by Thr466Ala-SHP2 (T/A-SHP2). Mutation of Thr466 does not cause significant structural changes in the active site as revealed by the trapping of the epidermal growth factor receptor (EGFR), the physiological substrate of SHP2, and by orthovanadate competition experiments. Based on these results and previous other works, we propose that the role of Thr466 in the catalytic process of SHP2 could be stabilizing the sulfhydryl group of Cys459 in its reduced state, a state that enables nucleophilic attack on the phosphate moiety of the substrate. The T/A-SHP2 harbors a single mutation and specifically interacts with the EGFR. Since the nucleophilic Cys459 and the proton donor Asp425 are intact in the T/A-SAHP2, it offers an excellent starting material for solving the structure of SHP2 in complex with its physiological substrate.

Ski negatively regulates erythroid differentiation through its interaction with GATA1.

The Ski oncoprotein dramatically affects cell growth, differentiation, and/or survival. Recently, Ski was shown to act in distinct signaling pathways including those involving nuclear receptors, transforming growth factor beta, and tumor suppressors. These divergent roles of Ski are probably dependent on Ski's capacity to bind multiple partners with disparate functions. In particular, Ski alters the growth and differentiation program of erythroid progenitor cells, leading to malignant leukemia. However, the mechanism underlying this important effect has remained elusive. Here we show that Ski interacts with GATA1, a transcription factor essential in erythropoiesis. Using a Ski mutant deficient in GATA1 binding, we show that this Ski-GATA1 interaction is critical for Ski's ability to repress GATA1-mediated transcription and block erythroid differentiation. Furthermore, the repression of GATA1-mediated transcription involves Ski's ability to block DNA binding of GATA1. This finding is in marked contrast to those in previous reports on the mechanism of repression by Ski, which have described a model involving the recruitment of corepressors into DNA-bound transcription complexes. We propose that Ski cooperates in the process of transformation in erythroid cells by interfering with GATA1 function, thereby contributing to erythroleukemia.

Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling.

The Src homology 2-containing phosphotyrosine phosphatase (SHP2) is primarily a positive effector of receptor tyrosine kinase signaling. However, the molecular mechanism by which SHP2 effects its biological function is unknown. In this report, we provide evidence that defines the molecular mechanism and site of action of SHP2 in the epidermal growth factor-induced mitogenic pathway. We demonstrate that SHP2 acts upstream of Ras and functions by increasing the half-life of activated Ras (GTP-Ras) in the cell by interfering with the process of Ras inactivation catalyzed by Ras GTPase-activating protein (RasGAP). It does so by inhibition of tyrosine phosphorylation-dependent translocation of RasGAP to the plasma membrane, to its substrate (GTP-Ras) microdomain. Inhibition is achieved through the dephosphorylation of RasGAP binding sites at the level of the plasma membrane. We have identified Tyr992 of the epidermal growth factor receptor (EGFR) to be one such site, since its mutation to Phe renders the EGFR refractory to the effect of dominant-negative SHP2. To our knowledge, this is the first report to outline the site and molecular mechanism of action of SHP2 in EGFR signaling, which may also serve as a model to describe its role in other receptor tyrosine kinase signaling pathways.

KRAS-dependent suppression of MYC enhances the sensitivity of cancer cells to cytotoxic agents.

KRAS is the most commonly mutated oncogene, frequently associated with some of the deadliest forms of cancer. However, the need for potent and specific KRAS inhibitors remains unmet. Here, we evaluated the effects of selected cytotoxic agents on oncogenic KRAS signaling and drug response. The data provided new insights into the functional interaction between the KRAS and MYC pathways and revealed key differences between WT and mutant KRAS expressing cells. Systematic investigation of non-small cell lung cancer cell lines revealed that KRAS mutation can paradoxically increase the sensitivity of cells to cytotoxic agents. We identify MYC as a key regulator of the cellular stress responses and tumor cell viability as MYC expression was suppressed in drug-sensitive but not resistant cells. Furthermore, this suppression was driven by hyperactive KRAS/MAPK signaling. Our findings support a direct link between MYC and cancer cell viability, and raise the possibility that inactivation of MYC may be an effective therapeutic strategy for KRAS mutant tumors across various cancer types.

The Ski oncoprotein is upregulated and localized at the centrosomes and mitotic spindle during mitosis.

Ski is an oncoprotein that represses transforming growth factor-beta and nuclear receptor signaling. Despite evidence that relates increased Ski protein levels directly with tumor progression in human cells, the signaling pathways that regulate Ski expression are mostly unidentified. Here we show that the Ski protein levels vary throughout the cell cycle, being lowest at G0/G1. This reduction in Ski protein levels results from proteosomal degradation as suggested by in vivo ubiquitination of Ski and the effects of proteosomal inhibitors. In contrast, an upregulation of the Ski protein was observed in cells going through mitosis. At this stage, we also found that Ski is phosphorylated. In vitro and in vivo data suggest that the phosphorylation of Ski in mitosis is carried out by the main kinase controlling the progression of mitosis, namely cdc2/cyclinB. Interestingly, immunofluorescence experiments, supported by biochemical data, show not only an increase in the Ski protein levels, but also a dramatic redistribution of Ski to the centrosomes and mitotic spindle throughout mitosis. Studies to date on Ski have focused on its role as a transcriptional regulator. However, Ski's increased level and specific relocalization during mitosis suggest that Ski might play a distinct role during this particular phase of the cell cycle.

An electrostatic engine model for autoinhibition and activation of the epidermal growth factor receptor (EGFR/ErbB) family.

We propose a new mechanism to explain autoinhibition of the epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases based on a structural model that postulates both their juxtamembrane and protein tyrosine kinase domains bind electrostatically to acidic lipids in the plasma membrane, restricting access of the kinase domain to substrate tyrosines. Ligand-induced dimerization promotes partial trans autophosphorylation of ErbB1, leading to a rapid rise in intracellular [Ca(2+)] that can activate calmodulin. We postulate the Ca(2+)/calmodulin complex binds rapidly to residues 645--660 of the juxtamembrane domain, reversing its net charge from +8 to -8 and repelling it from the negatively charged inner leaflet of the membrane. The repulsion has two consequences: it releases electrostatically sequestered phosphatidylinositol 4,5-bisphosphate (PIP(2)), and it disengages the kinase domain from the membrane, allowing it to become fully active and phosphorylate an adjacent ErbB molecule or other substrate. We tested various aspects of the model by measuring ErbB juxtamembrane peptide binding to phospholipid vesicles using both a centrifugation assay and fluorescence correlation spectroscopy; analyzing the kinetics of interactions between ErbB peptides, membranes, and Ca(2+)/calmodulin using fluorescence stop flow; assessing ErbB1 activation in Cos1 cells; measuring fluorescence resonance energy transfer between ErbB peptides and PIP(2); and making theoretical electrostatic calculations on atomic models of membranes and ErbB juxtamembrane and kinase domains.

Synthesis and Preclinical Evaluation of a Highly Improved Anticancer Prodrug Activated by Histone Deacetylases and Cathepsin L.

Lack of absolute selectivity against cancer cells is a major limitation for current cancer therapies. In the previous study, we developed a prodrug strategy for selective cancer therapy using a masked cytotoxic agent puromycin [Boc-Lys(Ac)-Puromycin], which can be sequentially activated by histone deacetylases (HDACs) and cathepsin L (CTSL) to kill cancer cells expressing high levels of both enzymes. Despite the promise as a selective cancer therapy, its requirement of relatively high dosage could be a potential issue in the clinical setting. To address this issue, we aimed to further improve the overall efficacy of our prodrug strategy. Since the proteolytic cleavage by CTSL is the rate-limiting step for the drug activation, we sought to improve the substrate structure for CTSL activity by modifying the α-amino protecting group of lysine. Here we show that protection with Fmoc [Fmoc-Lys(Ac)-Puromycin] exhibits a marked improvement in overall anticancer efficacy compared to the original Boc-Lys(Ac)-Puromycin and this is mainly due to the highly efficient cellular uptake besides its improved substrate structure. Furthermore, to address a concern that the improved drug efficacy might direct high toxicity to the normal cells, we confirmed that Fmoc-Lys(Ac)-Puromycin still retains excellent cancer selectivity in vitro and no obvious systemic off-target toxicity in vivo. Thus our preclinical evaluation data presented here demonstrate that the Fmoc-Lys(Ac)-Puromycin exhibits substantially improved anticancer efficacy, further supporting our approach for the selective cancer therapy.

A Novel EGFR Extracellular Domain Mutant, EGFRΔ768, Possesses Distinct Biological and Biochemical Properties in Neuroblastoma.

UNLABELLED: EGFR is a popular therapeutic target for many cancers. EGFR inhibitors have been tested in children with refractory neuroblastoma. Interestingly, partial response or stable disease was observed in a few neuroblastoma patients. As EGFR mutations are biomarkers for response to anti-EGFR drugs, primary neuroblastoma tumors and cell lines were screened for mutations. A novel EGFR extracellular domain deletion mutant, EGFRΔ768, was discovered and the biologic and biochemical properties of this mutant were characterized and compared with wild-type and EGFRvIII receptors. EGFRΔ768 was found to be constitutively active and localized to the cell surface. Its expression conferred resistance to etoposide and drove proliferation as well as invasion of cancer cells. While EGFRΔ768 had similarity to EGFRvIII, its biologic and biochemical properties were distinctly different from both the EGFRvIII and wild-type receptors. Even though erlotinib inhibited EGFRΔ768, its effect on the mutant was not as strong as that on wild-type EGFR and EGFRvIII. In addition, downstream signaling of EGFRΔ768 was different from that of the wild-type receptor. In conclusion, this is the first study to demonstrate that neuroblastoma express not only EGFRvIII, but also a novel EGFR extracellular domain deletion mutant, EGFRΔ768. The EGFRΔ768 also possesses distinct biologic and biochemical properties which might have therapeutic implications for neuroblastoma as well as other tumors expressing this novel mutant. IMPLICATIONS: Neuroblastoma expressed a novel EGFR mutant which possesses distinct biologic and biochemical properties that might have therapeutic implications. Mol Cancer Res; 14(8); 740-52. ©2016 AACR.