Dataset Information

Phosphoproteomics of induced and inhibited early Hh signaling in human medulloblastoma cells

ABSTRACT: Aberrant sonic hedeghog signaling is implicated in the development of various cancer entities such as medulloblastoma. The canonical signaling cascade has been studied for years. Activation of GLI transcription factors was revealed as the driving force upon pathway activation. Phosphorylation by Proteinkinase A, Casein Kinase 1 and Glycogen Synthase Kinase 3 β has been found to influence the degradation of the GLI transcription factors. However, the deeper role of phosphorylation in the signal transduction remains unclear. We, therefore, applied comprehensive HPLC-MS/MS based phosphoproteomics to reveal phosphorylation dynamics underlying the chemical activation and inhibition of sonic hedgehog signaling in human medulloblastoma cells. Human medulloblastoma cells were treated with SAG (Hh pathway induction) and Vismodegib (Hh pathway inhibition) for 5 and 15 minutes. Our phosphoproteomic profiling revealed a central role of phosphorylation in the regulation of ciliary assembly, trafficking and signal transduction after 5 minutes treatment. ERK/MAPK signaling besides protein kinase A signaling and mTOR signaling were differentially regulated. Activation of Polo-like kinase 1 and inhibtion of Caseinkinase 2A1 was characteristic for Vismodegib treatment while SAG treatment induced Aurora kinase A activity. Distinctive phosphorylation of central players of sonic Hh signaling such as Smoothened, SUFU, Gli2 and Gli3 was obtained after 15 minutes treatment.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Medulloblastoma,Brain Cancer

SUBMITTER: Tamara Scheidt

LAB HEAD: Christian G. Huber

PROVIDER: PXD013408 | Pride | 2021-09-08

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	15min_HILIC_final.mzTab	Mztab
	15min_HILIC_final_extractPH.csv	Csv
	15min_pH8_PQ_final.csv	Csv
	15min_pH8_final.mzTab	Mztab
	5min_HILIC_final.mzTab	Mztab

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Publications

Phosphoproteomics of short-term hedgehog signaling in human medulloblastoma cells.

Scheidt Tamara T Alka Oliver O Gonczarowska-Jorge Humberto H Gruber Wolfgang W Rathje Florian F Dell'Aica Margherita M Rurik Marc M Kohlbacher Oliver O Zahedi René P RP Aberger Fritz F Huber Christian G CG

Cell communication and signaling : CCS 20200623 1

<h4>Background</h4>Aberrant hedgehog (HH) signaling is implicated in the development of various cancer entities such as medulloblastoma. Activation of GLI transcription factors was revealed as the driving force upon pathway activation. Increased phosphorylation of essential effectors such as Smoothened (SMO) and GLI proteins by kinases including Protein Kinase A, Casein Kinase 1, and Glycogen Synthase Kinase 3 β controls effector activity, stability and processing. However, a deeper and more com ...[more]

PMID: 32576205

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Project description:Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor of many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling profoundly depend on interactions with other pathways such as epidermal growth factor receptor-mediated signaling which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. We demonstrate that the human medulloblastoma cell line Daoy possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic Hedgehog or Smoothened agonist induced expression of GLI1 protein and prevented processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed on the transcriptomic as well as proteomic level. Daoy cells responded to the co-treatment by downregulating GLI1, PTCH, and HHIP on the transcript level which was also seen when Amphiregulin (AREG) was used instead of EGF. The finding that EGFR signaling silences proteins acting as negative regulators of HH signaling is firstly described here as a novel crosstalk mechanism. Furthermore, combined EGFR/HH signaling maintains high GLI1 protein levels contrasting its downregulation on the transcript level. On the other hand, high level synergism was observed with respect to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile pointing to more wide-spread, yet context-dependent, interactions between HH/GLI and growth factor receptor signaling in human malignancies. To study interactions between HH- and EGF-induced signaling, time-resolved measurements were carried out over a period of 24 h at 14 different time points after stimulation by EGF with and without additional stimulation by SHH. Furthermore, as a control, cells without any stimulation by EGF and SHH (control) and cells in the presents of SHH were analyzed. Overall, three biological replicates of 60 different treatment/timepoints were analyzed yielding 180 different samples.

Project description:Mutations in Hedgehog (Hh) pathway genes, leading to constitutive activation of Smoothened (Smo), occur in sporadic medulloblastoma, the most common brain cancer in children. Antagonists of Smo induce tumor regression in mouse models of medulloblastoma and hold great promise for targeted therapy for this tumor. However, acquired resistance has emerged as one of the major challenges of targeted cancer therapy. Here, we describe novel mechanisms of acquired resistance to Smo antagonists in medulloblastoma. NVP-LDE225, a potent and selective Smo antagonist, inhibits Hh signaling and induces tumor regressions in allograft models of medulloblastoma that are driven by mutations of Patched (Ptch), a tumor suppressor in the Hh pathway. However, after long-term treatment, evidence of acquired resistance was observed. Genome-wide profiling of resistant tumors revealed distinct mechanisms to evade the inhibitory effects of Smo antagonists. Chromosomal amplification of Gli2, a downstream effector of Hh signaling, reactivated Hh signaling and restored tumor growth. Analysis of pathway gene-expression signatures selectively deregulated in resistant tumors identified increased phosphoinosite-3-kinase (PI3K) signaling as another potential resistance mechanism. Probing the functional relevance of increased PI3K signaling, we showed that the combination of NVP-LDE225 with the dual PI3K/mTOR inhibitor NVP-BEZ235 markedly delayed the development of resistance. Our findings have important clinical implications for future treatment strategies in medulloblastoma. mRNA profiling: RNA was prepared from tumours from vehicle or NVP-LDE225 treated nude mice allografted with medulloblastoma tumors derived from Ptch+/-p53-/- transgenic mouse and hybridized on Affymetrix Mouse Genome 430 2.0 RNA expression array. The dosage terminology (BID & QD) reflects the dosing schedule, where BID = twice a day, QD = once a day. aCGH: DNA was prepared from tumors from vehicle or NVP-LDE225 treated nude mice allografted with medulloblastoma tumors derived from Ptch+/-p53-/- transgenic mouse and hybridized on Agilent mouse CGH 244K Array.

Project description:Recent fate mapping studies concluded that EMT is not required for metastasis of carcinomas. Here we challenge this conclusion by showing that it failed to account for possible crosstalk between EMT and non-EMT cells that promotes dissemination of non-EMT cells. In breast cancer models, EMT cells induce increased metastasis of weakly metastatic, non-EMT tumor cells in a paracrine manner in part by non-cell autonomous activation of the GLI transcription factor. Treatment with GANT61, a GLI1/2 inhibitor, but not with IPI-926, a Smoothened inhibitor, blocks this effect, and inhibits growth in PDX models. In human breast tumors, the EMT-TFs strongly correlate with activated Hedgehog/GLI signaling, but not with the Hh ligands. Our findings indicate that EMT contributes to metastasis in part via non-cell autonomous effects that activate the Hh pathway. 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Because metastases in patients appear epithelial, the reverse process, mesenchymal-to-epithelial transition (MET), may occur to allow tumor cell colonization in secondary metastatic sites, establishing cellular plasticity as an important aspect of tumor progression. However, the role of EMT in carcinoma metastasis is controversial. Recent lineage tracing studies argue against the requirement of EMT for metastasis, as reporter-tagged cells that underwent a previous EMT were not found at the secondary site. However, these studies did not address the potential cooperation between EMT and non-EMT cells during the metastatic process, as EMT cancer cells may enable non-EMT cells to gain access to the secondary site, leading to macrometastatic growth. Thus, metastasis can be influenced by intratumoral heterogeneity: where a small proportion of primary tumor cells that have undergone an EMT may influence neighboring, non-EMT tumor cells. Twist1, Snail1, and Six1 are EMT-inducing transcription factors (EMT-TFs) that have all been associated with breast cancer metastasis. All three EMT-TFs regulate critical developmental processes such as cell survival, migration and invasion; in part by influencing EMT. In addition, they are downregulated post-embryogenesis, but re-expressed in various cancers where they cell autonomously induce EMT, resulting in increased percentages of tumor initiating cells (TICs) and enhanced metastasis. In carcinomas, Twist1 and Snail1 transcriptionally repress E-Cadherin (E- Cad) and upregulate mesenchymal genes. Similarly, Six1 overexpression induces EMT by regulating E-Cad localization and altering other EMT markers. During development and cancer, EMT-TFs act in concert with several signaling networks including TGFβ, Wnt, and Hedghog (Hh). 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