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The invadopodia scaffold protein Tks5 is required for the growth of human breast cancer cells in vitro and in vivo.

Blouw B, Patel M, Iizuka S, Abdullah C, You WK, Huang X, Li JL, Diaz B, Stallcup WB, Courtneidge SA - PLoS ONE (2015)

Bottom Line: We found that Tks5 is expressed to high levels in approximately 50% of primary invasive breast cancers.Knockdown of Tks5 expression in breast cancer cells resulted in decreased growth, both in 3D in vitro cultures and in vivo.Together, this work establishes an important role for Tks5 in tumor growth in vivo, and suggests that invadopodia may play broad roles in tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Tumor Microenvironment and Metastasis Program, NCI Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.

ABSTRACT
The ability of cancer cells to invade underlies metastatic progression. One mechanism by which cancer cells can become invasive is through the formation of structures called invadopodia, which are dynamic, actin-rich membrane protrusions that are sites of focal extracellular matrix degradation. While there is a growing consensus that invadopodia are instrumental in tumor metastasis, less is known about whether they are involved in tumor growth, particularly in vivo. The adaptor protein Tks5 is an obligate component of invadopodia, and is linked molecularly to both actin-remodeling proteins and pericellular proteases. Tks5 appears to localize exclusively to invadopodia in cancer cells, and in vitro studies have demonstrated its critical requirement for the invasive nature of these cells, making it an ideal surrogate to investigate the role of invadopodia in vivo. In this study, we examined how Tks5 contributes to human breast cancer progression. We used immunohistochemistry and RNA sequencing data to evaluate Tks5 expression in clinical samples, and we characterized the role of Tks5 in breast cancer progression using RNA interference and orthotopic implantation in SCID-Beige mice. We found that Tks5 is expressed to high levels in approximately 50% of primary invasive breast cancers. Furthermore, high expression was correlated with poor outcome, particularly in those patients with late relapse of stage I/II disease. Knockdown of Tks5 expression in breast cancer cells resulted in decreased growth, both in 3D in vitro cultures and in vivo. Moreover, our data also suggest that Tks5 is important for the integrity and permeability of the tumor vasculature. Together, this work establishes an important role for Tks5 in tumor growth in vivo, and suggests that invadopodia may play broad roles in tumor progression.

No MeSH data available.


Related in: MedlinePlus

Tks5 is required for tumor progression.MDA-MB-231-Luc cells were infected to stably express an inducible TetOn lentivirus where the levels of the Tks5 shRNA are under the control of the tetracycline promoter (see Materials and methods for the experimental procedure). A) Immunoblot demonstrating Tks5 expression reduction in the MDA-MB-231-Luc cell line in dose- and time-dependent fashion in response to doxycycline exposure. B) TetOn/D6 were injected orthotopically (blue, red, and green lines) compared to SCR and no doxycycline controls (grey and purple lines, respectively) under three conditions: when Tks5 was already reduced by in vitro exposure of the cells to doxycycline for 10 days (DOX A) (blue line), when unexposed cells were injected into the animal and the animal received doxycycline starting at the day of injection (DOX B) (red line), as well as when the animals received doxycycline in the drinking water for the first time after the tumor has been growing for 7 days (DOX C) (green line). C) Animals were given doxycycline 15 days after tumor cell injection and after randomization of the mice. TetOn/D6 mice were divided up in 3 groups where 2 groups received doxycycline in the drinking water at different time points and 1 group received doxycycline-free drinking water. Tumor volumes were measured at different time points as described in Materials and methods, and tumors were allowed to grow to a final volume of approximately 2cm3. N = 4 mice per tumor type. Experiments were performed in duplicate. D-I) Tumors from Fig 4C were analyzed for vascularization by CD31 (panel D-E), for apoptosis by TUNEL (panel F-G) and for proliferation using Ki-67 (panel H-I) immunofluorescence staining. Scale bar: 100μm. Images are representative for all experiments performed. J-K) Quantification of positive immunohistochemical and immunofluorescence staining. Graphs show immunopositive cells for apoptosis (TUNEL) (panel J) and proliferative (Ki67) (panel K) markers at the day of dissection (endpoint of experiment). Data were expressed as mean ± SD. One-way ANOVA or a Student’s t test was used to calculate p values.
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pone.0121003.g005: Tks5 is required for tumor progression.MDA-MB-231-Luc cells were infected to stably express an inducible TetOn lentivirus where the levels of the Tks5 shRNA are under the control of the tetracycline promoter (see Materials and methods for the experimental procedure). A) Immunoblot demonstrating Tks5 expression reduction in the MDA-MB-231-Luc cell line in dose- and time-dependent fashion in response to doxycycline exposure. B) TetOn/D6 were injected orthotopically (blue, red, and green lines) compared to SCR and no doxycycline controls (grey and purple lines, respectively) under three conditions: when Tks5 was already reduced by in vitro exposure of the cells to doxycycline for 10 days (DOX A) (blue line), when unexposed cells were injected into the animal and the animal received doxycycline starting at the day of injection (DOX B) (red line), as well as when the animals received doxycycline in the drinking water for the first time after the tumor has been growing for 7 days (DOX C) (green line). C) Animals were given doxycycline 15 days after tumor cell injection and after randomization of the mice. TetOn/D6 mice were divided up in 3 groups where 2 groups received doxycycline in the drinking water at different time points and 1 group received doxycycline-free drinking water. Tumor volumes were measured at different time points as described in Materials and methods, and tumors were allowed to grow to a final volume of approximately 2cm3. N = 4 mice per tumor type. Experiments were performed in duplicate. D-I) Tumors from Fig 4C were analyzed for vascularization by CD31 (panel D-E), for apoptosis by TUNEL (panel F-G) and for proliferation using Ki-67 (panel H-I) immunofluorescence staining. Scale bar: 100μm. Images are representative for all experiments performed. J-K) Quantification of positive immunohistochemical and immunofluorescence staining. Graphs show immunopositive cells for apoptosis (TUNEL) (panel J) and proliferative (Ki67) (panel K) markers at the day of dissection (endpoint of experiment). Data were expressed as mean ± SD. One-way ANOVA or a Student’s t test was used to calculate p values.

Mentions: The poor tumor growth we observed in Tks5-KD cells could reflect either a requirement for Tks5 expression for the establishment of mammary tumors, or an ongoing requirement for Tks5 for tumor growth. To distinguish between these possibilities, we next generated a mouse mammary tumor model in which human Tks5 expression was doxycycline-regulated. MDA-MB-231-Luc breast cancer cells were infected to stably express an inducible TetOn lentivirus where the levels of the Tks5 D6 shRNA are under the control of the doxycycline promoter [18]. Prior to in vivo studies, we confirmed via immunoblotting that Tks5 knockdown in the TetOn/D6 cells occurred in a dose- and time-dependent fashion in response to doxycycline exposure (Fig 5A). Then we established three test groups. Group 1 consisted of TetOn/D6 cells exposed to doxycycline for 10 days in vitro prior to orthotopic injection into mammary fat pads, with continued exposure in vivo (Fig 5B). In Group 2 the TetOn/D6 cells were not exposed to doxycycline in vitro, but doxycycline was added to the drinking water starting on the day of orthotopic injection (Fig 5B). For Group 3, animals injected with TetOn/D6 cells were exposed to doxycycline in the drinking water starting at day 7 post-injection, once tumors were established (Fig 5B). Knockdown of Tks5 impaired tumor growth to a similar extent under all 3 conditions, compared to the growth of TetOn/D6 cells which were never treated with doxycyline (p<0.05 by two-way repeated ANOVA with Bonferroni post-hoc test). (Fig 5B). In addition, the tumors in all three treated groups were significantly smaller than the tumors formed by TetOn/D6 cells which were never treated with doxycycline (p<0.01 by two-way repeated ANOVA with Bonferroni post-hoc test). Similar results were also obtained when doxycycline treatment was delayed until 15 days after injection (Fig 5C). We further analyzed the tumors from this experiment histologically. We observed decreased vessel density in doxycycline-regulated TetOn/D6 tumors compared to untreated TetOn/D6 cells tumors (Fig 5D and 5E), as well as increased apoptosis (Fig 5F-5G and 5J) and decreased cell proliferation (Fig 5H-5I and 5K). To summarize, these studies reveal that continued Tks5 expression is required for tumor growth.


The invadopodia scaffold protein Tks5 is required for the growth of human breast cancer cells in vitro and in vivo.

Blouw B, Patel M, Iizuka S, Abdullah C, You WK, Huang X, Li JL, Diaz B, Stallcup WB, Courtneidge SA - PLoS ONE (2015)

Tks5 is required for tumor progression.MDA-MB-231-Luc cells were infected to stably express an inducible TetOn lentivirus where the levels of the Tks5 shRNA are under the control of the tetracycline promoter (see Materials and methods for the experimental procedure). A) Immunoblot demonstrating Tks5 expression reduction in the MDA-MB-231-Luc cell line in dose- and time-dependent fashion in response to doxycycline exposure. B) TetOn/D6 were injected orthotopically (blue, red, and green lines) compared to SCR and no doxycycline controls (grey and purple lines, respectively) under three conditions: when Tks5 was already reduced by in vitro exposure of the cells to doxycycline for 10 days (DOX A) (blue line), when unexposed cells were injected into the animal and the animal received doxycycline starting at the day of injection (DOX B) (red line), as well as when the animals received doxycycline in the drinking water for the first time after the tumor has been growing for 7 days (DOX C) (green line). C) Animals were given doxycycline 15 days after tumor cell injection and after randomization of the mice. TetOn/D6 mice were divided up in 3 groups where 2 groups received doxycycline in the drinking water at different time points and 1 group received doxycycline-free drinking water. Tumor volumes were measured at different time points as described in Materials and methods, and tumors were allowed to grow to a final volume of approximately 2cm3. N = 4 mice per tumor type. Experiments were performed in duplicate. D-I) Tumors from Fig 4C were analyzed for vascularization by CD31 (panel D-E), for apoptosis by TUNEL (panel F-G) and for proliferation using Ki-67 (panel H-I) immunofluorescence staining. Scale bar: 100μm. Images are representative for all experiments performed. J-K) Quantification of positive immunohistochemical and immunofluorescence staining. Graphs show immunopositive cells for apoptosis (TUNEL) (panel J) and proliferative (Ki67) (panel K) markers at the day of dissection (endpoint of experiment). Data were expressed as mean ± SD. One-way ANOVA or a Student’s t test was used to calculate p values.
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Related In: Results  -  Collection

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Show All Figures
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pone.0121003.g005: Tks5 is required for tumor progression.MDA-MB-231-Luc cells were infected to stably express an inducible TetOn lentivirus where the levels of the Tks5 shRNA are under the control of the tetracycline promoter (see Materials and methods for the experimental procedure). A) Immunoblot demonstrating Tks5 expression reduction in the MDA-MB-231-Luc cell line in dose- and time-dependent fashion in response to doxycycline exposure. B) TetOn/D6 were injected orthotopically (blue, red, and green lines) compared to SCR and no doxycycline controls (grey and purple lines, respectively) under three conditions: when Tks5 was already reduced by in vitro exposure of the cells to doxycycline for 10 days (DOX A) (blue line), when unexposed cells were injected into the animal and the animal received doxycycline starting at the day of injection (DOX B) (red line), as well as when the animals received doxycycline in the drinking water for the first time after the tumor has been growing for 7 days (DOX C) (green line). C) Animals were given doxycycline 15 days after tumor cell injection and after randomization of the mice. TetOn/D6 mice were divided up in 3 groups where 2 groups received doxycycline in the drinking water at different time points and 1 group received doxycycline-free drinking water. Tumor volumes were measured at different time points as described in Materials and methods, and tumors were allowed to grow to a final volume of approximately 2cm3. N = 4 mice per tumor type. Experiments were performed in duplicate. D-I) Tumors from Fig 4C were analyzed for vascularization by CD31 (panel D-E), for apoptosis by TUNEL (panel F-G) and for proliferation using Ki-67 (panel H-I) immunofluorescence staining. Scale bar: 100μm. Images are representative for all experiments performed. J-K) Quantification of positive immunohistochemical and immunofluorescence staining. Graphs show immunopositive cells for apoptosis (TUNEL) (panel J) and proliferative (Ki67) (panel K) markers at the day of dissection (endpoint of experiment). Data were expressed as mean ± SD. One-way ANOVA or a Student’s t test was used to calculate p values.
Mentions: The poor tumor growth we observed in Tks5-KD cells could reflect either a requirement for Tks5 expression for the establishment of mammary tumors, or an ongoing requirement for Tks5 for tumor growth. To distinguish between these possibilities, we next generated a mouse mammary tumor model in which human Tks5 expression was doxycycline-regulated. MDA-MB-231-Luc breast cancer cells were infected to stably express an inducible TetOn lentivirus where the levels of the Tks5 D6 shRNA are under the control of the doxycycline promoter [18]. Prior to in vivo studies, we confirmed via immunoblotting that Tks5 knockdown in the TetOn/D6 cells occurred in a dose- and time-dependent fashion in response to doxycycline exposure (Fig 5A). Then we established three test groups. Group 1 consisted of TetOn/D6 cells exposed to doxycycline for 10 days in vitro prior to orthotopic injection into mammary fat pads, with continued exposure in vivo (Fig 5B). In Group 2 the TetOn/D6 cells were not exposed to doxycycline in vitro, but doxycycline was added to the drinking water starting on the day of orthotopic injection (Fig 5B). For Group 3, animals injected with TetOn/D6 cells were exposed to doxycycline in the drinking water starting at day 7 post-injection, once tumors were established (Fig 5B). Knockdown of Tks5 impaired tumor growth to a similar extent under all 3 conditions, compared to the growth of TetOn/D6 cells which were never treated with doxycyline (p<0.05 by two-way repeated ANOVA with Bonferroni post-hoc test). (Fig 5B). In addition, the tumors in all three treated groups were significantly smaller than the tumors formed by TetOn/D6 cells which were never treated with doxycycline (p<0.01 by two-way repeated ANOVA with Bonferroni post-hoc test). Similar results were also obtained when doxycycline treatment was delayed until 15 days after injection (Fig 5C). We further analyzed the tumors from this experiment histologically. We observed decreased vessel density in doxycycline-regulated TetOn/D6 tumors compared to untreated TetOn/D6 cells tumors (Fig 5D and 5E), as well as increased apoptosis (Fig 5F-5G and 5J) and decreased cell proliferation (Fig 5H-5I and 5K). To summarize, these studies reveal that continued Tks5 expression is required for tumor growth.

Bottom Line: We found that Tks5 is expressed to high levels in approximately 50% of primary invasive breast cancers.Knockdown of Tks5 expression in breast cancer cells resulted in decreased growth, both in 3D in vitro cultures and in vivo.Together, this work establishes an important role for Tks5 in tumor growth in vivo, and suggests that invadopodia may play broad roles in tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Tumor Microenvironment and Metastasis Program, NCI Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.

ABSTRACT
The ability of cancer cells to invade underlies metastatic progression. One mechanism by which cancer cells can become invasive is through the formation of structures called invadopodia, which are dynamic, actin-rich membrane protrusions that are sites of focal extracellular matrix degradation. While there is a growing consensus that invadopodia are instrumental in tumor metastasis, less is known about whether they are involved in tumor growth, particularly in vivo. The adaptor protein Tks5 is an obligate component of invadopodia, and is linked molecularly to both actin-remodeling proteins and pericellular proteases. Tks5 appears to localize exclusively to invadopodia in cancer cells, and in vitro studies have demonstrated its critical requirement for the invasive nature of these cells, making it an ideal surrogate to investigate the role of invadopodia in vivo. In this study, we examined how Tks5 contributes to human breast cancer progression. We used immunohistochemistry and RNA sequencing data to evaluate Tks5 expression in clinical samples, and we characterized the role of Tks5 in breast cancer progression using RNA interference and orthotopic implantation in SCID-Beige mice. We found that Tks5 is expressed to high levels in approximately 50% of primary invasive breast cancers. Furthermore, high expression was correlated with poor outcome, particularly in those patients with late relapse of stage I/II disease. Knockdown of Tks5 expression in breast cancer cells resulted in decreased growth, both in 3D in vitro cultures and in vivo. Moreover, our data also suggest that Tks5 is important for the integrity and permeability of the tumor vasculature. Together, this work establishes an important role for Tks5 in tumor growth in vivo, and suggests that invadopodia may play broad roles in tumor progression.

No MeSH data available.


Related in: MedlinePlus