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Wnt5a is strongly expressed at the leading edge in non-melanoma skin cancer, forming active gradients, while canonical Wnt signalling is repressed.

Pourreyron C, Reilly L, Proby C, Panteleyev A, Fleming C, McLean K, South AP, Foerster J - PLoS ONE (2012)

Bottom Line: Wnt5a is one of the so-called non-canonical Wnt ligands which do not act through β-catenin.In vitro migration assays show that Wnt5a concentration gradients determine its effect on keratinoctye migration: While chemotactic migration is inhibited by Wnt5a present in homogenous concentrations, it is enhanced in the presence of a Wnt5a gradient.This is confirmed by immunohistochemistry showing lack of nuclear β-catenin, as well as absent accumulation of Axin2.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Institute, College of Medicine, Dentistry, and Nursing, University of Dundee, Dundee, Scotland.

ABSTRACT
Wnt5a is one of the so-called non-canonical Wnt ligands which do not act through β-catenin. In normal development, Wnt5a is secreted and directs the migration of target cells along concentration gradients. The effect of Wnt5a on target cells is regulated by many factors, including the expression level of inhibitors and receptors. Dysregulated Wnt5a signalling facilitates invasion of multiple tumor types into adjacent tissue. However, the expression and distribution of Wnt5a in cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), as well as the effect of Wnt5a on keratinocyte migration has not been studied in detail to date. We here report that Wnt5a is upregulated in SCC and BCC and localised to the leading edge of tumors, as well as tumor-associated fibroblasts. The Wnt5a-triggered bundling of its receptor Fzd3 provides evidence of Wnt5a concentration gradients projecting into the tumor. In vitro migration assays show that Wnt5a concentration gradients determine its effect on keratinoctye migration: While chemotactic migration is inhibited by Wnt5a present in homogenous concentrations, it is enhanced in the presence of a Wnt5a gradient. Expression profiling of the Wnt pathway shows that the upregulation of Wnt5a in SCC is coupled to repression of canonical Wnt signalling. This is confirmed by immunohistochemistry showing lack of nuclear β-catenin, as well as absent accumulation of Axin2. Since both types of Wnt signalling act mutually antogonistically at multiple levels, the concurrent repression of canonical Wnt signalling suggests hyper-active Wnt5a signal transduction. Significantly, this combination of gene dysregulation is not observed in the benign hyperproliferative inflammatory skin disease psoriasis. Collectively, our data strongly suggest that Wnt5a signalling contributes to tissue invasion by non-melanoma skin cancer.

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Wnt5a inhibits keratinoctye migration when present in homogenous concentration, but acts as chemoattractant when present as gradient.A. Expression of endogenous and recombinant Wnt5a in whole cell lysates of stably transfected Wnt5a-overexpressing HaCat or control (HaCat-pcDNA) cells verified by western blot. B. Non-Wnt5a overexpressing HaCat-pcDNA cells were seeded in the upper chamber of a Transwell in 0.1% BSA DMEM in the absence or presence of recombinant Wnt5a at 1 µg/ml, as indicated in the figure. The lower chamber was filled with 600 µl DMEM containing 5% FCS as chemoattractant. Results are expressed as percentage of migrating cells when HaCat-pcDNA were seeded in 0.1% BSA DMEM only. The results shown represent mean ± s.d. of two independent experiment, each performed in triplicate, *p≤0.05. C. Comparison of Wnt5a-overexpressing and pcDNA control cell migration. Cells suspended in 0.1% BSA DMEM were seeded in the upper chamber. The lower chamber were filled with 600 µl DMEM containing 5% FCS as chemoattractant. Migration was assessed at 18 h using a colorimetric assay. Results are expressed as percentage of HaCat-pcDNA migrating cells. Results shown represent mean ± s.d. of n = 4 independent experiment, each performed in triplicate, *** p≤0.001. D. Scratch wound assay performed on mitomycin-C treated cells. During migration, HaCat-pcDNA (a, b, c), or Wnt5a-overexpressing cells (d, e, f) were maintained in DMEM containing 10% FCS. Pictures were taken just after the scratch was made (0 hrs) (a and d), as well as 18 h (b and e) and 24 h later (c and f). E. Migration of HaCat-pcDNA control cells in the presence of a Wnt5a concentration gradient. Wnt5a-overexpressing or pcDNA HaCat cells were seeded in the bottom wells of Transwell plates. Immediately before adding the inserts containing HaCat-pcDNA cells in the upper chamber, the media in the bottom wells was replaced to remove pre-secreted Wnt5a. Migration was assessed at 18 h. Results are expressed as percentage of HaCat-pcDNA migrating cells. Results shown represent mean ± s.d. of n = 3 independent experiments, each performed in triplicate, *** p≤0.001.
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pone-0031827-g006: Wnt5a inhibits keratinoctye migration when present in homogenous concentration, but acts as chemoattractant when present as gradient.A. Expression of endogenous and recombinant Wnt5a in whole cell lysates of stably transfected Wnt5a-overexpressing HaCat or control (HaCat-pcDNA) cells verified by western blot. B. Non-Wnt5a overexpressing HaCat-pcDNA cells were seeded in the upper chamber of a Transwell in 0.1% BSA DMEM in the absence or presence of recombinant Wnt5a at 1 µg/ml, as indicated in the figure. The lower chamber was filled with 600 µl DMEM containing 5% FCS as chemoattractant. Results are expressed as percentage of migrating cells when HaCat-pcDNA were seeded in 0.1% BSA DMEM only. The results shown represent mean ± s.d. of two independent experiment, each performed in triplicate, *p≤0.05. C. Comparison of Wnt5a-overexpressing and pcDNA control cell migration. Cells suspended in 0.1% BSA DMEM were seeded in the upper chamber. The lower chamber were filled with 600 µl DMEM containing 5% FCS as chemoattractant. Migration was assessed at 18 h using a colorimetric assay. Results are expressed as percentage of HaCat-pcDNA migrating cells. Results shown represent mean ± s.d. of n = 4 independent experiment, each performed in triplicate, *** p≤0.001. D. Scratch wound assay performed on mitomycin-C treated cells. During migration, HaCat-pcDNA (a, b, c), or Wnt5a-overexpressing cells (d, e, f) were maintained in DMEM containing 10% FCS. Pictures were taken just after the scratch was made (0 hrs) (a and d), as well as 18 h (b and e) and 24 h later (c and f). E. Migration of HaCat-pcDNA control cells in the presence of a Wnt5a concentration gradient. Wnt5a-overexpressing or pcDNA HaCat cells were seeded in the bottom wells of Transwell plates. Immediately before adding the inserts containing HaCat-pcDNA cells in the upper chamber, the media in the bottom wells was replaced to remove pre-secreted Wnt5a. Migration was assessed at 18 h. Results are expressed as percentage of HaCat-pcDNA migrating cells. Results shown represent mean ± s.d. of n = 3 independent experiments, each performed in triplicate, *** p≤0.001.

Mentions: The data summarised above suggested Wnt5a concentration gradients may be important for its effect on cell motility. To test this hypothesis functionally, we used human HaCat keratinocytes as a model. We stably transfected HaCat cells with a Wnt5a-expressing vector, or empty control vector (termed HaCat pcDNA). Expression of recombinant Wnt5a was verified by western-blot (fig. 6A). First, we assessed directed cell migration in a two-chamber Transwell assay. As shown in figure 6B, when recombinant Wnt5a was added directly to HaCat-pcDNA keratinocytes in the upper chamber, thus present in a homogenous concentration around migrating cells, it inhibited chemotactic migration toward 5% FCS present in the bottom well. Likewise, chemotactic migration was significantly reduced in cells overexpressing Wnt5a relative to non-overexpressing cells (fig. 6C). An analogous result was obtained in a short term (6 hours) migration assay using either 5% FCS or EGF as chemoattractant (fig. S2A). Moreover, in scratch assays, the migration of Wnt5a-overexpressing HaCat cells in 10% FCS DMEM (fig. 6D) toward the scratch edge was greatly reduced compared to HaCat-pcDNA cells (similar results were found when using 1% FCS as chemoattractant, fig. S2A). Thus, keratinocyte migration is inhibited when Wnt5a surrounds the cells at homogenous concentration. By contrast, when Wnt5a-secreting HaCat cells were seeded at the bottom of a Transwell chamber, thereby establishing an upward Wnt5a concentration gradient, migration of non-Wnt5a overexpressing Hacat cells seeded in the top chamber toward the Wnt5a source was significantly enhanced (fig. 6e). These data show that human keratinocytes are induced to migrate toward a Wnt5a-gradient, but that non-gradient Wnt5a decreases motility toward other chemoattractants.


Wnt5a is strongly expressed at the leading edge in non-melanoma skin cancer, forming active gradients, while canonical Wnt signalling is repressed.

Pourreyron C, Reilly L, Proby C, Panteleyev A, Fleming C, McLean K, South AP, Foerster J - PLoS ONE (2012)

Wnt5a inhibits keratinoctye migration when present in homogenous concentration, but acts as chemoattractant when present as gradient.A. Expression of endogenous and recombinant Wnt5a in whole cell lysates of stably transfected Wnt5a-overexpressing HaCat or control (HaCat-pcDNA) cells verified by western blot. B. Non-Wnt5a overexpressing HaCat-pcDNA cells were seeded in the upper chamber of a Transwell in 0.1% BSA DMEM in the absence or presence of recombinant Wnt5a at 1 µg/ml, as indicated in the figure. The lower chamber was filled with 600 µl DMEM containing 5% FCS as chemoattractant. Results are expressed as percentage of migrating cells when HaCat-pcDNA were seeded in 0.1% BSA DMEM only. The results shown represent mean ± s.d. of two independent experiment, each performed in triplicate, *p≤0.05. C. Comparison of Wnt5a-overexpressing and pcDNA control cell migration. Cells suspended in 0.1% BSA DMEM were seeded in the upper chamber. The lower chamber were filled with 600 µl DMEM containing 5% FCS as chemoattractant. Migration was assessed at 18 h using a colorimetric assay. Results are expressed as percentage of HaCat-pcDNA migrating cells. Results shown represent mean ± s.d. of n = 4 independent experiment, each performed in triplicate, *** p≤0.001. D. Scratch wound assay performed on mitomycin-C treated cells. During migration, HaCat-pcDNA (a, b, c), or Wnt5a-overexpressing cells (d, e, f) were maintained in DMEM containing 10% FCS. Pictures were taken just after the scratch was made (0 hrs) (a and d), as well as 18 h (b and e) and 24 h later (c and f). E. Migration of HaCat-pcDNA control cells in the presence of a Wnt5a concentration gradient. Wnt5a-overexpressing or pcDNA HaCat cells were seeded in the bottom wells of Transwell plates. Immediately before adding the inserts containing HaCat-pcDNA cells in the upper chamber, the media in the bottom wells was replaced to remove pre-secreted Wnt5a. Migration was assessed at 18 h. Results are expressed as percentage of HaCat-pcDNA migrating cells. Results shown represent mean ± s.d. of n = 3 independent experiments, each performed in triplicate, *** p≤0.001.
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pone-0031827-g006: Wnt5a inhibits keratinoctye migration when present in homogenous concentration, but acts as chemoattractant when present as gradient.A. Expression of endogenous and recombinant Wnt5a in whole cell lysates of stably transfected Wnt5a-overexpressing HaCat or control (HaCat-pcDNA) cells verified by western blot. B. Non-Wnt5a overexpressing HaCat-pcDNA cells were seeded in the upper chamber of a Transwell in 0.1% BSA DMEM in the absence or presence of recombinant Wnt5a at 1 µg/ml, as indicated in the figure. The lower chamber was filled with 600 µl DMEM containing 5% FCS as chemoattractant. Results are expressed as percentage of migrating cells when HaCat-pcDNA were seeded in 0.1% BSA DMEM only. The results shown represent mean ± s.d. of two independent experiment, each performed in triplicate, *p≤0.05. C. Comparison of Wnt5a-overexpressing and pcDNA control cell migration. Cells suspended in 0.1% BSA DMEM were seeded in the upper chamber. The lower chamber were filled with 600 µl DMEM containing 5% FCS as chemoattractant. Migration was assessed at 18 h using a colorimetric assay. Results are expressed as percentage of HaCat-pcDNA migrating cells. Results shown represent mean ± s.d. of n = 4 independent experiment, each performed in triplicate, *** p≤0.001. D. Scratch wound assay performed on mitomycin-C treated cells. During migration, HaCat-pcDNA (a, b, c), or Wnt5a-overexpressing cells (d, e, f) were maintained in DMEM containing 10% FCS. Pictures were taken just after the scratch was made (0 hrs) (a and d), as well as 18 h (b and e) and 24 h later (c and f). E. Migration of HaCat-pcDNA control cells in the presence of a Wnt5a concentration gradient. Wnt5a-overexpressing or pcDNA HaCat cells were seeded in the bottom wells of Transwell plates. Immediately before adding the inserts containing HaCat-pcDNA cells in the upper chamber, the media in the bottom wells was replaced to remove pre-secreted Wnt5a. Migration was assessed at 18 h. Results are expressed as percentage of HaCat-pcDNA migrating cells. Results shown represent mean ± s.d. of n = 3 independent experiments, each performed in triplicate, *** p≤0.001.
Mentions: The data summarised above suggested Wnt5a concentration gradients may be important for its effect on cell motility. To test this hypothesis functionally, we used human HaCat keratinocytes as a model. We stably transfected HaCat cells with a Wnt5a-expressing vector, or empty control vector (termed HaCat pcDNA). Expression of recombinant Wnt5a was verified by western-blot (fig. 6A). First, we assessed directed cell migration in a two-chamber Transwell assay. As shown in figure 6B, when recombinant Wnt5a was added directly to HaCat-pcDNA keratinocytes in the upper chamber, thus present in a homogenous concentration around migrating cells, it inhibited chemotactic migration toward 5% FCS present in the bottom well. Likewise, chemotactic migration was significantly reduced in cells overexpressing Wnt5a relative to non-overexpressing cells (fig. 6C). An analogous result was obtained in a short term (6 hours) migration assay using either 5% FCS or EGF as chemoattractant (fig. S2A). Moreover, in scratch assays, the migration of Wnt5a-overexpressing HaCat cells in 10% FCS DMEM (fig. 6D) toward the scratch edge was greatly reduced compared to HaCat-pcDNA cells (similar results were found when using 1% FCS as chemoattractant, fig. S2A). Thus, keratinocyte migration is inhibited when Wnt5a surrounds the cells at homogenous concentration. By contrast, when Wnt5a-secreting HaCat cells were seeded at the bottom of a Transwell chamber, thereby establishing an upward Wnt5a concentration gradient, migration of non-Wnt5a overexpressing Hacat cells seeded in the top chamber toward the Wnt5a source was significantly enhanced (fig. 6e). These data show that human keratinocytes are induced to migrate toward a Wnt5a-gradient, but that non-gradient Wnt5a decreases motility toward other chemoattractants.

Bottom Line: Wnt5a is one of the so-called non-canonical Wnt ligands which do not act through β-catenin.In vitro migration assays show that Wnt5a concentration gradients determine its effect on keratinoctye migration: While chemotactic migration is inhibited by Wnt5a present in homogenous concentrations, it is enhanced in the presence of a Wnt5a gradient.This is confirmed by immunohistochemistry showing lack of nuclear β-catenin, as well as absent accumulation of Axin2.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Institute, College of Medicine, Dentistry, and Nursing, University of Dundee, Dundee, Scotland.

ABSTRACT
Wnt5a is one of the so-called non-canonical Wnt ligands which do not act through β-catenin. In normal development, Wnt5a is secreted and directs the migration of target cells along concentration gradients. The effect of Wnt5a on target cells is regulated by many factors, including the expression level of inhibitors and receptors. Dysregulated Wnt5a signalling facilitates invasion of multiple tumor types into adjacent tissue. However, the expression and distribution of Wnt5a in cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), as well as the effect of Wnt5a on keratinocyte migration has not been studied in detail to date. We here report that Wnt5a is upregulated in SCC and BCC and localised to the leading edge of tumors, as well as tumor-associated fibroblasts. The Wnt5a-triggered bundling of its receptor Fzd3 provides evidence of Wnt5a concentration gradients projecting into the tumor. In vitro migration assays show that Wnt5a concentration gradients determine its effect on keratinoctye migration: While chemotactic migration is inhibited by Wnt5a present in homogenous concentrations, it is enhanced in the presence of a Wnt5a gradient. Expression profiling of the Wnt pathway shows that the upregulation of Wnt5a in SCC is coupled to repression of canonical Wnt signalling. This is confirmed by immunohistochemistry showing lack of nuclear β-catenin, as well as absent accumulation of Axin2. Since both types of Wnt signalling act mutually antogonistically at multiple levels, the concurrent repression of canonical Wnt signalling suggests hyper-active Wnt5a signal transduction. Significantly, this combination of gene dysregulation is not observed in the benign hyperproliferative inflammatory skin disease psoriasis. Collectively, our data strongly suggest that Wnt5a signalling contributes to tissue invasion by non-melanoma skin cancer.

Show MeSH
Related in: MedlinePlus