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Discoidin domain receptor 1 contributes to tumorigenesis through modulation of TGFBI expression.

Rudra-Ganguly N, Lowe C, Mattie M, Chang MS, Satpayev D, Verlinsky A, An Z, Hu L, Yang P, Challita-Eid P, Stover DR, Pereira DS - PLoS ONE (2014)

Bottom Line: Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis.Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression.The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Agensys Inc., an affiliate of Astellas Pharma Inc, Santa Monica, CA, United States of America.

ABSTRACT
Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFβ-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.

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TGFBI modulates growth and migration of BXPC3 cells.(a) Exogenous addition of TGFBI completely eliminated BXPC3 colonies as determined by crystal violet staining. (b) and (d) Wound healing capacity of BXPC3 cells was reduced by TGFBI in the absence and presence of collagen. The area of wound closure was photographed at different time points over a period of 40 hours. (c) and (e) The rate of wound healing over time is presented here for both conditions. The area closure was measured using the Image J program. TGFBI treatment showed an overall 50% inhibition of wound healing (***, p<0.0001). In all cases media supplemented with BSA was used as a negative control. BSA (▾), TGFBI (▽).
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pone-0111515-g005: TGFBI modulates growth and migration of BXPC3 cells.(a) Exogenous addition of TGFBI completely eliminated BXPC3 colonies as determined by crystal violet staining. (b) and (d) Wound healing capacity of BXPC3 cells was reduced by TGFBI in the absence and presence of collagen. The area of wound closure was photographed at different time points over a period of 40 hours. (c) and (e) The rate of wound healing over time is presented here for both conditions. The area closure was measured using the Image J program. TGFBI treatment showed an overall 50% inhibition of wound healing (***, p<0.0001). In all cases media supplemented with BSA was used as a negative control. BSA (▾), TGFBI (▽).

Mentions: We designed in vitro experiments to assess if exogenous TGFBI could recapitulate the phenotypes observed upon DDR1 silencing. BXPC3 cells were tested in both colony forming and wound healing assays in the presence of recombinant TGFBI with and without the addition of exogenous collagen. Based on a preliminary dose titration of TGFBI, we selected 50 µg/ml as the optimal concentration to use in functional assays that does not induce apoptosis (Fig. S4). Upon addition of recombinant TGFBI, we noticed very few colonies after 10 days both in the presence and absence of collagen (Fig. 5a). This robust growth inhibitory effect of TGFBI was similar to that observed upon DDR1 silencing. Fig. 5b to 5e show the effect of TGFBI on the wound healing process. At a longer incubation time of 40 hours, exogenous collagen shows modest inhibition of migration which is more pronounced in the presence of TGFBI. From the rate of wound healing calculated between 16 to 40 hours, recombinant TGFBI inhibited wound closure by 30%–50% compared to the control group (p = 0.0002). In the presence of collagen, TGFBI inhibited wound closure by 30–40% compared to the control collagen alone group (p = 0.0025). Under both conditions, TGFBI addition prevented wound closure at study termination.


Discoidin domain receptor 1 contributes to tumorigenesis through modulation of TGFBI expression.

Rudra-Ganguly N, Lowe C, Mattie M, Chang MS, Satpayev D, Verlinsky A, An Z, Hu L, Yang P, Challita-Eid P, Stover DR, Pereira DS - PLoS ONE (2014)

TGFBI modulates growth and migration of BXPC3 cells.(a) Exogenous addition of TGFBI completely eliminated BXPC3 colonies as determined by crystal violet staining. (b) and (d) Wound healing capacity of BXPC3 cells was reduced by TGFBI in the absence and presence of collagen. The area of wound closure was photographed at different time points over a period of 40 hours. (c) and (e) The rate of wound healing over time is presented here for both conditions. The area closure was measured using the Image J program. TGFBI treatment showed an overall 50% inhibition of wound healing (***, p<0.0001). In all cases media supplemented with BSA was used as a negative control. BSA (▾), TGFBI (▽).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4219757&req=5

pone-0111515-g005: TGFBI modulates growth and migration of BXPC3 cells.(a) Exogenous addition of TGFBI completely eliminated BXPC3 colonies as determined by crystal violet staining. (b) and (d) Wound healing capacity of BXPC3 cells was reduced by TGFBI in the absence and presence of collagen. The area of wound closure was photographed at different time points over a period of 40 hours. (c) and (e) The rate of wound healing over time is presented here for both conditions. The area closure was measured using the Image J program. TGFBI treatment showed an overall 50% inhibition of wound healing (***, p<0.0001). In all cases media supplemented with BSA was used as a negative control. BSA (▾), TGFBI (▽).
Mentions: We designed in vitro experiments to assess if exogenous TGFBI could recapitulate the phenotypes observed upon DDR1 silencing. BXPC3 cells were tested in both colony forming and wound healing assays in the presence of recombinant TGFBI with and without the addition of exogenous collagen. Based on a preliminary dose titration of TGFBI, we selected 50 µg/ml as the optimal concentration to use in functional assays that does not induce apoptosis (Fig. S4). Upon addition of recombinant TGFBI, we noticed very few colonies after 10 days both in the presence and absence of collagen (Fig. 5a). This robust growth inhibitory effect of TGFBI was similar to that observed upon DDR1 silencing. Fig. 5b to 5e show the effect of TGFBI on the wound healing process. At a longer incubation time of 40 hours, exogenous collagen shows modest inhibition of migration which is more pronounced in the presence of TGFBI. From the rate of wound healing calculated between 16 to 40 hours, recombinant TGFBI inhibited wound closure by 30%–50% compared to the control group (p = 0.0002). In the presence of collagen, TGFBI inhibited wound closure by 30–40% compared to the control collagen alone group (p = 0.0025). Under both conditions, TGFBI addition prevented wound closure at study termination.

Bottom Line: Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis.Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression.The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Agensys Inc., an affiliate of Astellas Pharma Inc, Santa Monica, CA, United States of America.

ABSTRACT
Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFβ-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.

Show MeSH
Related in: MedlinePlus