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Loss of Endocan tumorigenic properties after alternative splicing of exon 2.

Depontieu F, Grigoriu BD, Scherpereel A, Adam E, Delehedde M, Gosset P, Lassalle P - BMC Cancer (2008)

Bottom Line: Endocan expression confers tumorigenic properties to epithelial cell lines or accelerate the growth of already tumorigenic cells.Our results showed that the exon 2 deletion impairs synthesis of the glycan chain, known to be involved in the pro-tumoral effect of endocan.Our results emphasize the key role of the polypeptide sequence encoded by the exon 2 of endocan gene in tumorigenesis, and suggest that this sequence could be a target for future therapies against cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U774, Lille 59019, France. fdepont2@jhmi.edu

ABSTRACT

Background: Endocan was originally described as a dermatan sulfate proteoglycan found freely circulating in the blood. Endocan expression confers tumorigenic properties to epithelial cell lines or accelerate the growth of already tumorigenic cells. This molecule is the product of a single gene composed of 3 exons. Previous data showed that endocan mRNA is subject to alternative splicing with possible generation of two protein products. In the present study we identified, and functionally characterized, the alternative spliced product of the endocan gene: the exon 2-deleted endocan, called endocanDelta2.

Methods: Stable, endocanDelta2-overexpressing cell lines were generated to investigate the biological activities of this new alternatively spliced product of endocan gene. Tumorigenesis was studied by inoculating endocan and endocanDelta2 expressing cell lines subcutaneously in SCID mice. Biochemical properties of endocan and endocanDelta2 were studied after production of recombinant proteins in various cell lines of human and murine origin.

Results: Our results showed that the exon 2 deletion impairs synthesis of the glycan chain, known to be involved in the pro-tumoral effect of endocan. EndocanDelta2 did not promote tumor formation by 293 cells implanted in the skin of severe combined immunodeficient (SCID) mice.

Conclusion: Our results emphasize the key role of the polypeptide sequence encoded by the exon 2 of endocan gene in tumorigenesis, and suggest that this sequence could be a target for future therapies against cancer.

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A: DEAE-sepharose binding property of endocan and endocanΔ2. The crude supernatants (Input) of transiently transfected HEK 293 cells with endocan (grey box) or endocanΔ2 (white box) (n = 4). The bound materials were eluted in 1 M NaCl (Eluate). Endocan and endocanΔ2 levels were measured by ELISA (median values with error bars showing the range). B: The eluates from DEAE-Sepharose column were digested overnight with chondroitinase (Ch) ABC. Digested (lanes 2, 4, 6, 8) and undigested (lanes 1, 3, 5, 7) samples were analyzed under reducing (lanes 1–4) or nonreducing (lanes 5–8) conditions. Black arrow indicates the endocan translation product and white arrow the endocanΔ2 translation product. Stars indicate the multimeric forms of endocanΔ2. C: Western blot analysis of endocan immunoprecipitated from endothelial or transfected cells supernatants. Lane 1: HUVEC; lane 2: endocan-HEK 293; lane 3: endocanΔ2-HEK 293. Black arrow indicates the endocan and endocanΔ2 translation products. Star indicate the multimeric form of endocanΔ2; Arrowheads indicate the non-specific bands originating from the secondary Ab. D. Endocan (grey box) and endocanΔ2 (white box) levels in the supernatant and cell lysate of HEK293 cells after 72 hours of culture assessed by ELISA (median, with error bars showing the range, n = 6) **, p = 0.022.
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Figure 4: A: DEAE-sepharose binding property of endocan and endocanΔ2. The crude supernatants (Input) of transiently transfected HEK 293 cells with endocan (grey box) or endocanΔ2 (white box) (n = 4). The bound materials were eluted in 1 M NaCl (Eluate). Endocan and endocanΔ2 levels were measured by ELISA (median values with error bars showing the range). B: The eluates from DEAE-Sepharose column were digested overnight with chondroitinase (Ch) ABC. Digested (lanes 2, 4, 6, 8) and undigested (lanes 1, 3, 5, 7) samples were analyzed under reducing (lanes 1–4) or nonreducing (lanes 5–8) conditions. Black arrow indicates the endocan translation product and white arrow the endocanΔ2 translation product. Stars indicate the multimeric forms of endocanΔ2. C: Western blot analysis of endocan immunoprecipitated from endothelial or transfected cells supernatants. Lane 1: HUVEC; lane 2: endocan-HEK 293; lane 3: endocanΔ2-HEK 293. Black arrow indicates the endocan and endocanΔ2 translation products. Star indicate the multimeric form of endocanΔ2; Arrowheads indicate the non-specific bands originating from the secondary Ab. D. Endocan (grey box) and endocanΔ2 (white box) levels in the supernatant and cell lysate of HEK293 cells after 72 hours of culture assessed by ELISA (median, with error bars showing the range, n = 6) **, p = 0.022.

Mentions: The glycan moiety of endocan is known to play important role in tumorigenesis [17]. We thus enquired if the glycanation of endocanΔ2 is modified compared to that of endocan. By its glycan, human endocan binds to the DEAE-Sepharose. The same property was observed for endocanΔ2 (Fig. 4A). The DEAE-bound endocanΔ2 was detected at 40 kDa in reducing conditions (Fig. 4B, lane 3). Treatment with chondroitinase ABC induced a shift from 40 to 14 kDa (Fig. 4B, lane 4), confirming that endocanΔ2, as well as endocan, is a proteoglycan with a chondroitin/dermatan sulfate chain. Surprisingly, not all endocanΔ2 is glycanated and 47.4 % (ranging from 42.9 % to 50.2 %) of endocanΔ2 was recovered in the flow-through of DEAE-Sepharose (Fig. 4A). This was confirmed using crude supernatants in non reducing conditions revealing two additional bands at 14 and 26 kDa below the glycanated endocanΔ2 spanning from 35 to 70 kDa (Fig. 4C, lane 3). Thus, the deletion of the exon 2-derived sequence impairs the glycanation of the endocanΔ2 polypeptide, partially explaining the lack of tumorigenic activity.


Loss of Endocan tumorigenic properties after alternative splicing of exon 2.

Depontieu F, Grigoriu BD, Scherpereel A, Adam E, Delehedde M, Gosset P, Lassalle P - BMC Cancer (2008)

A: DEAE-sepharose binding property of endocan and endocanΔ2. The crude supernatants (Input) of transiently transfected HEK 293 cells with endocan (grey box) or endocanΔ2 (white box) (n = 4). The bound materials were eluted in 1 M NaCl (Eluate). Endocan and endocanΔ2 levels were measured by ELISA (median values with error bars showing the range). B: The eluates from DEAE-Sepharose column were digested overnight with chondroitinase (Ch) ABC. Digested (lanes 2, 4, 6, 8) and undigested (lanes 1, 3, 5, 7) samples were analyzed under reducing (lanes 1–4) or nonreducing (lanes 5–8) conditions. Black arrow indicates the endocan translation product and white arrow the endocanΔ2 translation product. Stars indicate the multimeric forms of endocanΔ2. C: Western blot analysis of endocan immunoprecipitated from endothelial or transfected cells supernatants. Lane 1: HUVEC; lane 2: endocan-HEK 293; lane 3: endocanΔ2-HEK 293. Black arrow indicates the endocan and endocanΔ2 translation products. Star indicate the multimeric form of endocanΔ2; Arrowheads indicate the non-specific bands originating from the secondary Ab. D. Endocan (grey box) and endocanΔ2 (white box) levels in the supernatant and cell lysate of HEK293 cells after 72 hours of culture assessed by ELISA (median, with error bars showing the range, n = 6) **, p = 0.022.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254430&req=5

Figure 4: A: DEAE-sepharose binding property of endocan and endocanΔ2. The crude supernatants (Input) of transiently transfected HEK 293 cells with endocan (grey box) or endocanΔ2 (white box) (n = 4). The bound materials were eluted in 1 M NaCl (Eluate). Endocan and endocanΔ2 levels were measured by ELISA (median values with error bars showing the range). B: The eluates from DEAE-Sepharose column were digested overnight with chondroitinase (Ch) ABC. Digested (lanes 2, 4, 6, 8) and undigested (lanes 1, 3, 5, 7) samples were analyzed under reducing (lanes 1–4) or nonreducing (lanes 5–8) conditions. Black arrow indicates the endocan translation product and white arrow the endocanΔ2 translation product. Stars indicate the multimeric forms of endocanΔ2. C: Western blot analysis of endocan immunoprecipitated from endothelial or transfected cells supernatants. Lane 1: HUVEC; lane 2: endocan-HEK 293; lane 3: endocanΔ2-HEK 293. Black arrow indicates the endocan and endocanΔ2 translation products. Star indicate the multimeric form of endocanΔ2; Arrowheads indicate the non-specific bands originating from the secondary Ab. D. Endocan (grey box) and endocanΔ2 (white box) levels in the supernatant and cell lysate of HEK293 cells after 72 hours of culture assessed by ELISA (median, with error bars showing the range, n = 6) **, p = 0.022.
Mentions: The glycan moiety of endocan is known to play important role in tumorigenesis [17]. We thus enquired if the glycanation of endocanΔ2 is modified compared to that of endocan. By its glycan, human endocan binds to the DEAE-Sepharose. The same property was observed for endocanΔ2 (Fig. 4A). The DEAE-bound endocanΔ2 was detected at 40 kDa in reducing conditions (Fig. 4B, lane 3). Treatment with chondroitinase ABC induced a shift from 40 to 14 kDa (Fig. 4B, lane 4), confirming that endocanΔ2, as well as endocan, is a proteoglycan with a chondroitin/dermatan sulfate chain. Surprisingly, not all endocanΔ2 is glycanated and 47.4 % (ranging from 42.9 % to 50.2 %) of endocanΔ2 was recovered in the flow-through of DEAE-Sepharose (Fig. 4A). This was confirmed using crude supernatants in non reducing conditions revealing two additional bands at 14 and 26 kDa below the glycanated endocanΔ2 spanning from 35 to 70 kDa (Fig. 4C, lane 3). Thus, the deletion of the exon 2-derived sequence impairs the glycanation of the endocanΔ2 polypeptide, partially explaining the lack of tumorigenic activity.

Bottom Line: Endocan expression confers tumorigenic properties to epithelial cell lines or accelerate the growth of already tumorigenic cells.Our results showed that the exon 2 deletion impairs synthesis of the glycan chain, known to be involved in the pro-tumoral effect of endocan.Our results emphasize the key role of the polypeptide sequence encoded by the exon 2 of endocan gene in tumorigenesis, and suggest that this sequence could be a target for future therapies against cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U774, Lille 59019, France. fdepont2@jhmi.edu

ABSTRACT

Background: Endocan was originally described as a dermatan sulfate proteoglycan found freely circulating in the blood. Endocan expression confers tumorigenic properties to epithelial cell lines or accelerate the growth of already tumorigenic cells. This molecule is the product of a single gene composed of 3 exons. Previous data showed that endocan mRNA is subject to alternative splicing with possible generation of two protein products. In the present study we identified, and functionally characterized, the alternative spliced product of the endocan gene: the exon 2-deleted endocan, called endocanDelta2.

Methods: Stable, endocanDelta2-overexpressing cell lines were generated to investigate the biological activities of this new alternatively spliced product of endocan gene. Tumorigenesis was studied by inoculating endocan and endocanDelta2 expressing cell lines subcutaneously in SCID mice. Biochemical properties of endocan and endocanDelta2 were studied after production of recombinant proteins in various cell lines of human and murine origin.

Results: Our results showed that the exon 2 deletion impairs synthesis of the glycan chain, known to be involved in the pro-tumoral effect of endocan. EndocanDelta2 did not promote tumor formation by 293 cells implanted in the skin of severe combined immunodeficient (SCID) mice.

Conclusion: Our results emphasize the key role of the polypeptide sequence encoded by the exon 2 of endocan gene in tumorigenesis, and suggest that this sequence could be a target for future therapies against cancer.

Show MeSH
Related in: MedlinePlus