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. 2013;8(2):e56162.
doi: 10.1371/journal.pone.0056162. Epub 2013 Feb 13.

PBOV1 is a human de novo gene with tumor-specific expression that is associated with a positive clinical outcome of cancer

Nikolay Samusik  1 , Larisa Krukovskaya, Irina Meln, Evgeny Shilov, Andrey P Kozlov
Affiliations

PBOV1 is a human de novo gene with tumor-specific expression that is associated with a positive clinical outcome of cancer

Nikolay Samusik et al. PLoS One. 2013.

Abstract

PBOV1 is a known human protein-coding gene with an uncharacterized function. We have previously found that PBOV1 lacks orthologs in non-primate genomes and is expressed in a wide range of tumor types. Here we report that PBOV1 protein-coding sequence is human-specific and has originated de novo in the primate evolution through a series of frame-shift and stop codon mutations. We profiled PBOV1 expression in multiple cancer and normal tissue samples and found that it was expressed in 19 out of 34 tumors of various origins but completely lacked expression in any of the normal adult or fetal human tissues. We found that, unlike the cancer/testis antigens that are typically controlled by CpG island-containing promoters, PBOV1 was expressed from a GC-poor TATA-containing promoter which was not influenced by CpG demethylation and was inactive in testis. Our analysis of public microarray data suggests that PBOV1 activation in tumors could be dependent on the Hedgehog signaling pathway. Despite the recent de novo origin and the lack of identifiable functional signatures, a missense SNP in the PBOV1 coding sequence has been previously associated with an increased risk of breast cancer. Using publicly available microarray datasets, we found that high levels of PBOV1 expression in breast cancer and glioma samples were significantly associated with a positive outcome of the cancer disease. We also found that PBOV1 was highly expressed in primary but not in recurrent high-grade gliomas, suggesting the presence of a negative selection against PBOV1-expressing cancer cells. Our findings could contribute to the understanding of the mechanisms behind de novo gene origin and the possible role of tumors in this process.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Comparative genomics analysis reveals the de novo origin of the PBOV1 protein-coding sequence.
A: The evolutionary tree of 34 mammals with available genomic sequences. The values next to species names show fractions of CDS of human PBOV1 that could be aligned with the respective genome and fractions of encoded proteins (assuming that they exist) that could be aligned with the human PBOV1 protein. For selected taxons, the most probable values of those fractions in the last common ancestor (LCA) are given. The genome of LCA of Boreoeutheria most likely contained the start codon of PBOV1, 97% of respective genomic sequence (as the maximum of 97% of human sequence could be aligned to the genomes of horse and megabat) and 7% of the putative protein sequence. However, in rodents and Lagomorpha the frame was lost due to a mutation in the ATG codon. Laurasiatheria retain up to 97% of the genomic sequence homologous to PBOV1 CDS, but the protein homology is below 3% due to a synapomorphic frame-shift deletion. All higher primates contain at least 99% of human genomic sequence, but the protein homology is only 20%. An important evolutionary event along the human lineage was the A→T substitution at the position 90 in the last common ancestor of Hominidae which removed the stop codon. However, all Hominidae genomes lack an in-frame stop codon over the span of the human transcript, which could make the transcript in this species a target of the non-stop decay . Finally, a single nucleotide deletion that occurred after the divergence from chimp led to a frame-shift that finally shaped the modern human PBOV1 protein sequence. B: Multiple alignments of human PBOV1 CDS with orthologous loci from selected mammalian species. The stretches of genomes that contribute to the putative protein homology to human PBOV1 are highlighted in yellow, followed by the features that disrupt protein homology (frame-shifts and stop codons). For the sake of representation, the exact sequences of species-specific insertions are omitted from the alignment.
Figure 2
Figure 2. Expression profiling of PBOV1 and GAPDH (positive control) measured by PCR in cDNA panels from human normal tissues shows the lack of PBOV1 expression in adult and fetal normal tissures.
A. Human MTC Panel I (1–8), Human MTC Panel II (9–16): 1 – brain, 2 – heart, 3 – kidney, 4 – liver, 5 – lung, 6 – pancreas, 7 – placenta, 8 – skeletal muscle, 9 – colon, 10 – ovary, 11 – peripheral blood leukocyte, 12 – prostate, 13 – small intestine, 14 – spleen, 15 – testis, 16 – thymus; Full size images of gels are shown on Figure S1 and Figure S2 in File S1. B. Human Digestive System MTC Panel: 1 – cecum, 2 – colon, ascending 3 – colon, descending 4 – colon, transverse 5 – duodenum, 6 – esophagus, 7 – ileocecum, 8 – ileum, 9 – jejunum, 10 – liver, 11 – rectum, 12 – stomach. Full-sized images of gels are presented on Figure S5 and Figure S6 in File S1. C. Human Immune System MTC Panel (1–7), Human Fetal MTC Panel(8–15): 1 – bone marrow, 2 – fetal liver, 3 – lymph node, 4 – peripheral blood leukocyte, 5 – spleen, 6 – thymus, 7 – tonsil, 8 – fetal brain, 9 – fetal heart, 10 – fetal kidney, 11 – fetal liver, 12 – fetal lung, 13 – fetal skeletal muscle, 14 – fetal spleen, 15 – fetal thymus; A–C: NC – PCR with no template, PC – PCR with human DNA. Full size images of gels are shown on Figure S3 and Figure S4 in File S1.
Figure 3
Figure 3. PBOV1 expression profiling by PCR in cDNA panels from human tumors shows that PBOV1 is expressed in multiple tumor types.
A. Tumor cDNA Panel (BioChain Institute, USA): 1 – Brain medulloblastoma, with glioma, 2 – Lung squamous cell carcinoma, 3 – Kidney granular cell carcinoma, 4 – Kidney clear cell carcinoma, 5 – Liver cholangiocellular carcinoma, 6 – Hepatocellular carcinoma, 7 – Gallbladder adenocarcinoma, 8 – Esophagus squamous cell carcinoma, 9 – Stomach signet ring cell carcinoma, 10 – Small Intestine adenocarcinoma, 11 – Colon papillary adenocarcinoma, 12 – Rectum adenocarcinoma, 13 – Breast fibroadenoma, 14 – Ovary serous cystoadenocarcinoma, 15 – Fallopian tube medullary carcinoma, 16 – Uterus adenocarcinoma, 17 – Ureter papillary transitional cell carcinoma, 18 – Bladder transitional cell carcinoma, 19 – Testis seminoma, 20 – Prostate adenocarcinoma, 21 – Malignant melanoma, 22 – Skeletal Muscle malignancy fibrous histocytoma, 23 – Adrenal pheochromocytoma, 24 – Non-Hodgkin's lymphoma, 25 – Thyroid papillary adenocarcinoma, 26 – Parotid mixed tumor, 27 – Pancreas adenocarcinoma, 28 – Thymus seminoma, 29 – Spleen serous adenocarcinoma, 30 – Hodgkin's lymphoma, 31 – T cell Hodgkin's lymphoma, 32 – Malignant lymphoma. NC – PCR with no template, PC – PCR with human DNA. DNA contamination was controlled using gDNA-CTR primers. Full-sized images of gels are presented on Figure S7 and Figure S8 in File S1. B. PBOV1 expression in clinical tumor samples (see Materials and Methods for full description of samples). PBOV1 is expressed in breast cancer (9–250), ovary cancer (1, 6), cervical cancer (2, 13), endometrial cancer (156, 270), lung cancer (12, 14, 17), seminoma (7), meningioma (63), non-Hodgkin lymphomas (67, 82, 92, 102, 113) Full-sized images of gels are presented on Figure S9 and Figure S10 in File S1.
Figure 4
Figure 4. PBOV1 expression in breast cancer and proneural glioma is correlated to a positive clinical outcome of the disease.
A. Kaplan-Meier analysis of a pooled dataset of breast cancer expression profiles from six independent clinical studies shows that higher levels of PBOV1 expression positively correlated to relapse-free survival in breast cancer. Among clinical subgroups the effect was mostly pronounced in cases of lymph node positive cancers and in cases of grade 2 tumors (data obtained from GOBO online tool [34]). B. PBOV1 expression levels in clinical samples of estrogen receptor-positive breast cancer positively correlate to the patient relapse-free survival over 5 years following tamoxifen therapy (data obtained from GEO dataset GDS806 [35]). Error bars represent standard error of the mean. C. PBOV1 expression levels in clinical tumor samples from proneural glioma patients positively correlate with survival over 209 weeks (data obtained from GEO dataset GDS1816 [36]). Error bars represent standard error of the mean. D. Primary proneural gliomas have significantly higher expression levels of PBOV1 expression than recurrent ones (data obtained from GEO dataset GDS1816 [36]). Error bars represent standard error of the mean.
Figure 5
Figure 5. PBOV1 expression in pancreas cancer xenografts is downregulated by HhAntag treatment (data from GSE11981 dataset).
The data comes from a study that profiled the gene expression response of human pancreatic cancer xenografts in mice to the treatment with HhAntag, a potent inhibitor of Hedgehog signaling and a prospective anti-cancer drug . In three out of four replicates PBOV1 expression was downregulated by more than 75%.
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This work was funded by the Biomedical Centre and by the Russian-Belorussian program #K-32-NIR/111-3. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, except for Prof. Andrey P. Kozlov who, being the head of the Biomedical Centre, simultaneously authorized the funding and supervised this work.

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