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USP1 deubiquitinase: cellular functions, regulatory mechanisms and emerging potential as target in cancer therapy.

García-Santisteban I, Peters GJ, Giovannetti E, Rodríguez JA - Mol. Cancer (2013)

Bottom Line: Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy.We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data.Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Leioa, Spain.

ABSTRACT
Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the best-characterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.

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USP1 gene mutations and altered USP1 mRNA expression in lung cancer. A. Shematic representation of USP1 protein showing the position of 13 lung cancer-associated USP1 mutations reported to date (5 April 2013) in the COSMIC mutation database. Amino acid changes are indicated using the one-letter code. B. Relative USP1 mRNA expression normalized to GAPDH in paired samples of tumor tissue and adjacent normal tissue from four NSCLC patients (represented by different symbols), showing higher USP1 expression in tumor tissue. Total RNA was isolated using Trizol (Invitrogen), and complementary DNA (cDNA) was synthesized using the Dynamo cDNA synthesis kit for qRT-PCR (Thermo Scientific). Quantitative real-time PCR (qRT-PCR) was performed using the TaqMan Universal Master Mix (Applied Biosystems) according to the manufacturer’s instructions on an ABI Prism 7500 instrument. Assay-on-Demand Gene expression primers and probes (Applied Biosystems) were used to specifically amplify USP1 (Hs00163427_m1) and Human GAPD (GAPDH) Endogenous Control (4326317E). C. Graph comparing the relative expression of USP1 mRNA normalized to GAPDH internal control in normal lung tissue (white bar; four individual samples) with the expression in pooled samples of the three different NSCLC histological subtypes (grey bar; adenocarcinoma, squamous cell carcinoma and large cell carcinoma). Each pooled sample included laser-microdissected specimens from five different patients, as described previously [88]. qRT-PCR analysis was carried out as described above. Graph bars show mean ± SEM. USP1 was significantly overexpressed in the pooled tumor samples (***P < 0.001). D. Relative USP1 mRNA expression normalized to GAPDH in 20 different NSCLC cell lines compared to the average of four normal lung tissue mRNA samples. qRT-PCR analysis was carried out as described above. The relative mRNA levels were determined with the ΔΔCt method using the “normal” value as a reference. Graph bars show mean ± SD of two replicates.
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Figure 3: USP1 gene mutations and altered USP1 mRNA expression in lung cancer. A. Shematic representation of USP1 protein showing the position of 13 lung cancer-associated USP1 mutations reported to date (5 April 2013) in the COSMIC mutation database. Amino acid changes are indicated using the one-letter code. B. Relative USP1 mRNA expression normalized to GAPDH in paired samples of tumor tissue and adjacent normal tissue from four NSCLC patients (represented by different symbols), showing higher USP1 expression in tumor tissue. Total RNA was isolated using Trizol (Invitrogen), and complementary DNA (cDNA) was synthesized using the Dynamo cDNA synthesis kit for qRT-PCR (Thermo Scientific). Quantitative real-time PCR (qRT-PCR) was performed using the TaqMan Universal Master Mix (Applied Biosystems) according to the manufacturer’s instructions on an ABI Prism 7500 instrument. Assay-on-Demand Gene expression primers and probes (Applied Biosystems) were used to specifically amplify USP1 (Hs00163427_m1) and Human GAPD (GAPDH) Endogenous Control (4326317E). C. Graph comparing the relative expression of USP1 mRNA normalized to GAPDH internal control in normal lung tissue (white bar; four individual samples) with the expression in pooled samples of the three different NSCLC histological subtypes (grey bar; adenocarcinoma, squamous cell carcinoma and large cell carcinoma). Each pooled sample included laser-microdissected specimens from five different patients, as described previously [88]. qRT-PCR analysis was carried out as described above. Graph bars show mean ± SEM. USP1 was significantly overexpressed in the pooled tumor samples (***P < 0.001). D. Relative USP1 mRNA expression normalized to GAPDH in 20 different NSCLC cell lines compared to the average of four normal lung tissue mRNA samples. qRT-PCR analysis was carried out as described above. The relative mRNA levels were determined with the ΔΔCt method using the “normal” value as a reference. Graph bars show mean ± SD of two replicates.

Mentions: Mutational alteration of human DUBs, including USP1, does not appear to be a frequent event in cancer [30]. With the exception of CYLD, mutated in the familial cylindromatosis syndrome [85], and BAP1, mutated in several malignancies [86], no other DUBs have been reported to be recurrently mutated in specific tumor types. Nevertheless, whole genome analysis of cancer samples is uncovering low-frequency DUB mutations in human tumors. In the case of USP1, a survey of the COSMIC mutation database [87] revealed a total of 23 non-synonymous mutations in different tumor types. Thirteen of these mutations have been detected in lung cancer samples. Figure 3A shows the distribution of these lung cancer-associated mutations on USP1 protein. Most of these are missense mutations, resulting in amino acid substitutions, but the functional consequences of these protein changes remain to be established.


USP1 deubiquitinase: cellular functions, regulatory mechanisms and emerging potential as target in cancer therapy.

García-Santisteban I, Peters GJ, Giovannetti E, Rodríguez JA - Mol. Cancer (2013)

USP1 gene mutations and altered USP1 mRNA expression in lung cancer. A. Shematic representation of USP1 protein showing the position of 13 lung cancer-associated USP1 mutations reported to date (5 April 2013) in the COSMIC mutation database. Amino acid changes are indicated using the one-letter code. B. Relative USP1 mRNA expression normalized to GAPDH in paired samples of tumor tissue and adjacent normal tissue from four NSCLC patients (represented by different symbols), showing higher USP1 expression in tumor tissue. Total RNA was isolated using Trizol (Invitrogen), and complementary DNA (cDNA) was synthesized using the Dynamo cDNA synthesis kit for qRT-PCR (Thermo Scientific). Quantitative real-time PCR (qRT-PCR) was performed using the TaqMan Universal Master Mix (Applied Biosystems) according to the manufacturer’s instructions on an ABI Prism 7500 instrument. Assay-on-Demand Gene expression primers and probes (Applied Biosystems) were used to specifically amplify USP1 (Hs00163427_m1) and Human GAPD (GAPDH) Endogenous Control (4326317E). C. Graph comparing the relative expression of USP1 mRNA normalized to GAPDH internal control in normal lung tissue (white bar; four individual samples) with the expression in pooled samples of the three different NSCLC histological subtypes (grey bar; adenocarcinoma, squamous cell carcinoma and large cell carcinoma). Each pooled sample included laser-microdissected specimens from five different patients, as described previously [88]. qRT-PCR analysis was carried out as described above. Graph bars show mean ± SEM. USP1 was significantly overexpressed in the pooled tumor samples (***P < 0.001). D. Relative USP1 mRNA expression normalized to GAPDH in 20 different NSCLC cell lines compared to the average of four normal lung tissue mRNA samples. qRT-PCR analysis was carried out as described above. The relative mRNA levels were determined with the ΔΔCt method using the “normal” value as a reference. Graph bars show mean ± SD of two replicates.
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Figure 3: USP1 gene mutations and altered USP1 mRNA expression in lung cancer. A. Shematic representation of USP1 protein showing the position of 13 lung cancer-associated USP1 mutations reported to date (5 April 2013) in the COSMIC mutation database. Amino acid changes are indicated using the one-letter code. B. Relative USP1 mRNA expression normalized to GAPDH in paired samples of tumor tissue and adjacent normal tissue from four NSCLC patients (represented by different symbols), showing higher USP1 expression in tumor tissue. Total RNA was isolated using Trizol (Invitrogen), and complementary DNA (cDNA) was synthesized using the Dynamo cDNA synthesis kit for qRT-PCR (Thermo Scientific). Quantitative real-time PCR (qRT-PCR) was performed using the TaqMan Universal Master Mix (Applied Biosystems) according to the manufacturer’s instructions on an ABI Prism 7500 instrument. Assay-on-Demand Gene expression primers and probes (Applied Biosystems) were used to specifically amplify USP1 (Hs00163427_m1) and Human GAPD (GAPDH) Endogenous Control (4326317E). C. Graph comparing the relative expression of USP1 mRNA normalized to GAPDH internal control in normal lung tissue (white bar; four individual samples) with the expression in pooled samples of the three different NSCLC histological subtypes (grey bar; adenocarcinoma, squamous cell carcinoma and large cell carcinoma). Each pooled sample included laser-microdissected specimens from five different patients, as described previously [88]. qRT-PCR analysis was carried out as described above. Graph bars show mean ± SEM. USP1 was significantly overexpressed in the pooled tumor samples (***P < 0.001). D. Relative USP1 mRNA expression normalized to GAPDH in 20 different NSCLC cell lines compared to the average of four normal lung tissue mRNA samples. qRT-PCR analysis was carried out as described above. The relative mRNA levels were determined with the ΔΔCt method using the “normal” value as a reference. Graph bars show mean ± SD of two replicates.
Mentions: Mutational alteration of human DUBs, including USP1, does not appear to be a frequent event in cancer [30]. With the exception of CYLD, mutated in the familial cylindromatosis syndrome [85], and BAP1, mutated in several malignancies [86], no other DUBs have been reported to be recurrently mutated in specific tumor types. Nevertheless, whole genome analysis of cancer samples is uncovering low-frequency DUB mutations in human tumors. In the case of USP1, a survey of the COSMIC mutation database [87] revealed a total of 23 non-synonymous mutations in different tumor types. Thirteen of these mutations have been detected in lung cancer samples. Figure 3A shows the distribution of these lung cancer-associated mutations on USP1 protein. Most of these are missense mutations, resulting in amino acid substitutions, but the functional consequences of these protein changes remain to be established.

Bottom Line: Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy.We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data.Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Leioa, Spain.

ABSTRACT
Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the best-characterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.

Show MeSH
Related in: MedlinePlus