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Molecular mechanisms of activating c-MET in KSHV+ primary effusion lymphoma

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ABSTRACT

The oncogenic Kaposi's sarcoma–associated herpesvirus (KSHV) is a principal causative agent of primary effusion lymphoma (PEL), which is mostly seen in immunosuppressed patients. PEL is a rapidly progressing malignancy with a median survival time of approximately 6 months even under the conventional chemotherapy. We recently report that the hepatocyte growth factor (HGF)/c-MET pathway is highly activated in PEL cells and represents a promising therapeutic target (Blood. 2015;126(26):2821-31). However, the underlying mechanisms of c-MET activation within PEL cells remain largely unknown. To solve this puzzle, here we have utilized the next generation sequencing (NGS) based bioinformatics approach to investigate the genomic landscape of the c-MET gene and we found that there's no single nucleotide variations (SNVs) occurred in the c-MET genomic regions in a cohort of PEL samples. Consistently, Sanger sequencing analysis of frequently mutated exons such as exon 10, 14 and 19 shows no mutation of these c-MET exons in PEL cell-lines, which further supports the notion that mutations are not the major mechanism responsible for c-MET activation in PEL. Further, we found that a transmembrane receptor protein, Plexin-B1, is expressed in PEL cell-lines, which is required for c-MET-mediated PEL cell survival via direct protein interaction.

No MeSH data available.


Sequencing and alignment analysis of representative exons of c-MET from PEL cell-linesA-C. PCR products of exon 10, 14 and 19 of c-MET amplified from five PEL cell-lines were purified and sequenced as described in the Methods. The alignment analysis was performed using Vector NTI v10.0 software (Invitrogen), and the human c-MET sequence (GenBank #J02958) used as the reference sequence.
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Figure 4: Sequencing and alignment analysis of representative exons of c-MET from PEL cell-linesA-C. PCR products of exon 10, 14 and 19 of c-MET amplified from five PEL cell-lines were purified and sequenced as described in the Methods. The alignment analysis was performed using Vector NTI v10.0 software (Invitrogen), and the human c-MET sequence (GenBank #J02958) used as the reference sequence.

Mentions: To further confirm our SNVs analysis results, we performed Sanger sequencing of exon 10, 14 and 19 of c-MET, all of which have been reported frequent mutations in solid tumors as mentioned above. Our PCR results showed all the 5 examined KSHV+ PEL cell lines (BC-1, BC-3, BCP-1, BCBL-1 and JSC-1) having the same length of exon 10, 14 and 19 as that from A549, one of NSCLC cell-lines without c-MET missense mutations [20] (Figure 3). The Sanger sequencing of these c-MET exons PCR products and alignment analysis confirmed no detectable mutations within all the 5 PEL cell-lines, when compared to the human c-MET reference sequence (GenBank #J02958) (Figure 4). Taken together, our data indicate that mutations are not the major molecular mechanism responsible for c-MET activation in PEL tumor cells.


Molecular mechanisms of activating c-MET in KSHV+ primary effusion lymphoma
Sequencing and alignment analysis of representative exons of c-MET from PEL cell-linesA-C. PCR products of exon 10, 14 and 19 of c-MET amplified from five PEL cell-lines were purified and sequenced as described in the Methods. The alignment analysis was performed using Vector NTI v10.0 software (Invitrogen), and the human c-MET sequence (GenBank #J02958) used as the reference sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5392335&req=5

Figure 4: Sequencing and alignment analysis of representative exons of c-MET from PEL cell-linesA-C. PCR products of exon 10, 14 and 19 of c-MET amplified from five PEL cell-lines were purified and sequenced as described in the Methods. The alignment analysis was performed using Vector NTI v10.0 software (Invitrogen), and the human c-MET sequence (GenBank #J02958) used as the reference sequence.
Mentions: To further confirm our SNVs analysis results, we performed Sanger sequencing of exon 10, 14 and 19 of c-MET, all of which have been reported frequent mutations in solid tumors as mentioned above. Our PCR results showed all the 5 examined KSHV+ PEL cell lines (BC-1, BC-3, BCP-1, BCBL-1 and JSC-1) having the same length of exon 10, 14 and 19 as that from A549, one of NSCLC cell-lines without c-MET missense mutations [20] (Figure 3). The Sanger sequencing of these c-MET exons PCR products and alignment analysis confirmed no detectable mutations within all the 5 PEL cell-lines, when compared to the human c-MET reference sequence (GenBank #J02958) (Figure 4). Taken together, our data indicate that mutations are not the major molecular mechanism responsible for c-MET activation in PEL tumor cells.

View Article: PubMed Central - PubMed

ABSTRACT

The oncogenic Kaposi's sarcoma–associated herpesvirus (KSHV) is a principal causative agent of primary effusion lymphoma (PEL), which is mostly seen in immunosuppressed patients. PEL is a rapidly progressing malignancy with a median survival time of approximately 6 months even under the conventional chemotherapy. We recently report that the hepatocyte growth factor (HGF)/c-MET pathway is highly activated in PEL cells and represents a promising therapeutic target (Blood. 2015;126(26):2821-31). However, the underlying mechanisms of c-MET activation within PEL cells remain largely unknown. To solve this puzzle, here we have utilized the next generation sequencing (NGS) based bioinformatics approach to investigate the genomic landscape of the c-MET gene and we found that there's no single nucleotide variations (SNVs) occurred in the c-MET genomic regions in a cohort of PEL samples. Consistently, Sanger sequencing analysis of frequently mutated exons such as exon 10, 14 and 19 shows no mutation of these c-MET exons in PEL cell-lines, which further supports the notion that mutations are not the major mechanism responsible for c-MET activation in PEL. Further, we found that a transmembrane receptor protein, Plexin-B1, is expressed in PEL cell-lines, which is required for c-MET-mediated PEL cell survival via direct protein interaction.

No MeSH data available.