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The UPF1 RNA surveillance gene is commonly mutated in pancreatic adenosquamous carcinoma.

Liu C, Karam R, Zhou Y, Su F, Ji Y, Li G, Xu G, Lu L, Wang C, Song M, Zhu J, Wang Y, Zhao Y, Foo WC, Zuo M, Valasek MA, Javle M, Wilkinson MF, Lu Y - Nat. Med. (2014)

Bottom Line: Pancreatic adenosquamous carcinoma (ASC) is an enigmatic and aggressive tumor that has a worse prognosis and higher metastatic potential than its adenocarcinoma counterpart.These tumor-specific mutations alter UPF1 RNA splicing and perturb NMD, leading to upregulated levels of NMD substrate mRNAs.UPF1 mutations are, to our knowledge, the first known unique molecular signatures of pancreatic ASC.

View Article: PubMed Central - PubMed

Affiliation: 1] Clinical and Translational Cancer Research Center, The Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China. [2] Tongji University School of Life Science and Technology, Shanghai, China. [3].

ABSTRACT
Pancreatic adenosquamous carcinoma (ASC) is an enigmatic and aggressive tumor that has a worse prognosis and higher metastatic potential than its adenocarcinoma counterpart. Here we report that ASC tumors frequently harbor somatically acquired mutations in the UPF1 gene, which encodes the core component of the nonsense-mediated RNA decay (NMD) pathway. These tumor-specific mutations alter UPF1 RNA splicing and perturb NMD, leading to upregulated levels of NMD substrate mRNAs. UPF1 mutations are, to our knowledge, the first known unique molecular signatures of pancreatic ASC.

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ASC-specific UPF1 mutations trigger alternative UPF1 RNA splicing. (a) The indicated region of human UPF1 (nt 22,779-23,570, RefSeq accession number NC_000019.9) was cloned into the NdeI site of the pTBNde mini-gene construct. All mutations were generated by site-directed mutagenesis to match those in the indicated patient’s tumors. (b) The indicated region of human UPF1 (nt 33,138-34,490) was cloned and mutated as described in panel a. (c) RT-PCR analysis of HEK293 cells transfected with the constructs shown in panel a (primer locations are indicated by the arrows). Direct sequencing of the large (792 nt) and small (239 nt) bands indicated that they correspond to normally spliced and exon-skipped transcripts, respectively. The numbers below the gel are the average values from five independent transfections. (d) RT-PCR analysis performed as in panel c. Direct sequencing of the bands in the gel indicated they were derived from mRNA spliced in the manner shown in the schematic. The bands corresponding to normally spliced, alt 1, and alt 2 mRNA had lengths of 742, 1117, and 619 nt, respectively. The numbers below the gel are the average values from five independent transfections. (e) RT-PCR analysis of normal pancreas (NP) and ASC frozen samples (TU) from patients 15 and 16. RT-PCR sequencing results are indicated as schematics next to the gels. (f) Immunohistochemical analysis of UPF1 staining in ASC tumor samples from patient 15. Tumor (T) and normal tissue (NT) are indicated. Bottom panel shows H & E staining, with arrows pointing to the adenocarinoma component in this ASC tumor. The scale bar represents 200 micrometers on each picture.
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Figure 2: ASC-specific UPF1 mutations trigger alternative UPF1 RNA splicing. (a) The indicated region of human UPF1 (nt 22,779-23,570, RefSeq accession number NC_000019.9) was cloned into the NdeI site of the pTBNde mini-gene construct. All mutations were generated by site-directed mutagenesis to match those in the indicated patient’s tumors. (b) The indicated region of human UPF1 (nt 33,138-34,490) was cloned and mutated as described in panel a. (c) RT-PCR analysis of HEK293 cells transfected with the constructs shown in panel a (primer locations are indicated by the arrows). Direct sequencing of the large (792 nt) and small (239 nt) bands indicated that they correspond to normally spliced and exon-skipped transcripts, respectively. The numbers below the gel are the average values from five independent transfections. (d) RT-PCR analysis performed as in panel c. Direct sequencing of the bands in the gel indicated they were derived from mRNA spliced in the manner shown in the schematic. The bands corresponding to normally spliced, alt 1, and alt 2 mRNA had lengths of 742, 1117, and 619 nt, respectively. The numbers below the gel are the average values from five independent transfections. (e) RT-PCR analysis of normal pancreas (NP) and ASC frozen samples (TU) from patients 15 and 16. RT-PCR sequencing results are indicated as schematics next to the gels. (f) Immunohistochemical analysis of UPF1 staining in ASC tumor samples from patient 15. Tumor (T) and normal tissue (NT) are indicated. Bottom panel shows H & E staining, with arrows pointing to the adenocarinoma component in this ASC tumor. The scale bar represents 200 micrometers on each picture.

Mentions: The point mutations in the ASC tumors clustered in two regions of the UPF1 gene (Fig. 1b). Surprisingly, these mutations were nearly equally distributed in the exons and introns in these two regions. This raised the possibility that they alter UPF1 splicing by disrupting intronic splicing enhancers (ISEs) and exonic splicing enhancers (ESEs), which are essential for the inclusion of a subset of exons during splicing8,9. In support of this, a subset of the mutations in the ASC tumors disrupted predicted ESEs/ISEs (Fig. 1b and Supplementary Fig. 2). To directly test whether the ASC mutations disrupt UPF1 splicing, we generated UPF1 mini-gene constructs corresponding to the two regions mutated in ASC tumors (Fig. 2a,b). We then introduced, by site-specific mutagenesis, the mutations found in 15 of the patients. Transfection analysis showed that the wild-type versions of the two mini-gene constructs expressed normally spliced mRNA that included all exons, as determined by direct sequencing of the bands generated by RT-PCR (Fig. 2c,d). No other bands were detected, indicating that most of the mature mRNA generated by these mini-gene constructs was normally spliced. In contrast, all 15 mutant mini-genes expressed alternatively spliced transcripts (Fig. 2,d). While none of the patient’s mutations completely eliminated normal splicing, the ratio of alternatively spliced-to-normally spliced mRNA was 10 to 1 or higher in many cases. As confirmation, analysis of endogenous UPF1 splicing in 2 of 2 frozen tumor samples revealed an alternatively spliced UPF1 mRNA that was not present in the adjacent normal tissue (Fig. 2e). We conclude that ASC-specific mutations in the UPF1 gene trigger alternative splicing of UPF1 pre-mRNA.


The UPF1 RNA surveillance gene is commonly mutated in pancreatic adenosquamous carcinoma.

Liu C, Karam R, Zhou Y, Su F, Ji Y, Li G, Xu G, Lu L, Wang C, Song M, Zhu J, Wang Y, Zhao Y, Foo WC, Zuo M, Valasek MA, Javle M, Wilkinson MF, Lu Y - Nat. Med. (2014)

ASC-specific UPF1 mutations trigger alternative UPF1 RNA splicing. (a) The indicated region of human UPF1 (nt 22,779-23,570, RefSeq accession number NC_000019.9) was cloned into the NdeI site of the pTBNde mini-gene construct. All mutations were generated by site-directed mutagenesis to match those in the indicated patient’s tumors. (b) The indicated region of human UPF1 (nt 33,138-34,490) was cloned and mutated as described in panel a. (c) RT-PCR analysis of HEK293 cells transfected with the constructs shown in panel a (primer locations are indicated by the arrows). Direct sequencing of the large (792 nt) and small (239 nt) bands indicated that they correspond to normally spliced and exon-skipped transcripts, respectively. The numbers below the gel are the average values from five independent transfections. (d) RT-PCR analysis performed as in panel c. Direct sequencing of the bands in the gel indicated they were derived from mRNA spliced in the manner shown in the schematic. The bands corresponding to normally spliced, alt 1, and alt 2 mRNA had lengths of 742, 1117, and 619 nt, respectively. The numbers below the gel are the average values from five independent transfections. (e) RT-PCR analysis of normal pancreas (NP) and ASC frozen samples (TU) from patients 15 and 16. RT-PCR sequencing results are indicated as schematics next to the gels. (f) Immunohistochemical analysis of UPF1 staining in ASC tumor samples from patient 15. Tumor (T) and normal tissue (NT) are indicated. Bottom panel shows H & E staining, with arrows pointing to the adenocarinoma component in this ASC tumor. The scale bar represents 200 micrometers on each picture.
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Figure 2: ASC-specific UPF1 mutations trigger alternative UPF1 RNA splicing. (a) The indicated region of human UPF1 (nt 22,779-23,570, RefSeq accession number NC_000019.9) was cloned into the NdeI site of the pTBNde mini-gene construct. All mutations were generated by site-directed mutagenesis to match those in the indicated patient’s tumors. (b) The indicated region of human UPF1 (nt 33,138-34,490) was cloned and mutated as described in panel a. (c) RT-PCR analysis of HEK293 cells transfected with the constructs shown in panel a (primer locations are indicated by the arrows). Direct sequencing of the large (792 nt) and small (239 nt) bands indicated that they correspond to normally spliced and exon-skipped transcripts, respectively. The numbers below the gel are the average values from five independent transfections. (d) RT-PCR analysis performed as in panel c. Direct sequencing of the bands in the gel indicated they were derived from mRNA spliced in the manner shown in the schematic. The bands corresponding to normally spliced, alt 1, and alt 2 mRNA had lengths of 742, 1117, and 619 nt, respectively. The numbers below the gel are the average values from five independent transfections. (e) RT-PCR analysis of normal pancreas (NP) and ASC frozen samples (TU) from patients 15 and 16. RT-PCR sequencing results are indicated as schematics next to the gels. (f) Immunohistochemical analysis of UPF1 staining in ASC tumor samples from patient 15. Tumor (T) and normal tissue (NT) are indicated. Bottom panel shows H & E staining, with arrows pointing to the adenocarinoma component in this ASC tumor. The scale bar represents 200 micrometers on each picture.
Mentions: The point mutations in the ASC tumors clustered in two regions of the UPF1 gene (Fig. 1b). Surprisingly, these mutations were nearly equally distributed in the exons and introns in these two regions. This raised the possibility that they alter UPF1 splicing by disrupting intronic splicing enhancers (ISEs) and exonic splicing enhancers (ESEs), which are essential for the inclusion of a subset of exons during splicing8,9. In support of this, a subset of the mutations in the ASC tumors disrupted predicted ESEs/ISEs (Fig. 1b and Supplementary Fig. 2). To directly test whether the ASC mutations disrupt UPF1 splicing, we generated UPF1 mini-gene constructs corresponding to the two regions mutated in ASC tumors (Fig. 2a,b). We then introduced, by site-specific mutagenesis, the mutations found in 15 of the patients. Transfection analysis showed that the wild-type versions of the two mini-gene constructs expressed normally spliced mRNA that included all exons, as determined by direct sequencing of the bands generated by RT-PCR (Fig. 2c,d). No other bands were detected, indicating that most of the mature mRNA generated by these mini-gene constructs was normally spliced. In contrast, all 15 mutant mini-genes expressed alternatively spliced transcripts (Fig. 2,d). While none of the patient’s mutations completely eliminated normal splicing, the ratio of alternatively spliced-to-normally spliced mRNA was 10 to 1 or higher in many cases. As confirmation, analysis of endogenous UPF1 splicing in 2 of 2 frozen tumor samples revealed an alternatively spliced UPF1 mRNA that was not present in the adjacent normal tissue (Fig. 2e). We conclude that ASC-specific mutations in the UPF1 gene trigger alternative splicing of UPF1 pre-mRNA.

Bottom Line: Pancreatic adenosquamous carcinoma (ASC) is an enigmatic and aggressive tumor that has a worse prognosis and higher metastatic potential than its adenocarcinoma counterpart.These tumor-specific mutations alter UPF1 RNA splicing and perturb NMD, leading to upregulated levels of NMD substrate mRNAs.UPF1 mutations are, to our knowledge, the first known unique molecular signatures of pancreatic ASC.

View Article: PubMed Central - PubMed

Affiliation: 1] Clinical and Translational Cancer Research Center, The Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China. [2] Tongji University School of Life Science and Technology, Shanghai, China. [3].

ABSTRACT
Pancreatic adenosquamous carcinoma (ASC) is an enigmatic and aggressive tumor that has a worse prognosis and higher metastatic potential than its adenocarcinoma counterpart. Here we report that ASC tumors frequently harbor somatically acquired mutations in the UPF1 gene, which encodes the core component of the nonsense-mediated RNA decay (NMD) pathway. These tumor-specific mutations alter UPF1 RNA splicing and perturb NMD, leading to upregulated levels of NMD substrate mRNAs. UPF1 mutations are, to our knowledge, the first known unique molecular signatures of pancreatic ASC.

Show MeSH
Related in: MedlinePlus