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Spontaneous tumour regression in keratoacanthomas is driven by Wnt/retinoic acid signalling cross-talk.

Zito G, Saotome I, Liu Z, Ferro EG, Sun TY, Nguyen DX, Bilguvar K, Ko CJ, Greco V - Nat Commun (2014)

Bottom Line: A fundamental goal in cancer biology is to identify the cells and signalling pathways that are keys to induce tumour regression.Furthermore, we demonstrate that developmental programs utilized for skin hair follicle regeneration, such as Wnt, are hijacked to sustain tumour growth and that the retinoic acid (RA) signalling pathway promotes tumour regression by inhibiting Wnt signalling.Finally, we find that RA signalling can induce regression of malignant tumours that do not normally spontaneously regress, such as squamous cell carcinomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Yale Stem Cell Center, Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut 06510, USA.

ABSTRACT
A fundamental goal in cancer biology is to identify the cells and signalling pathways that are keys to induce tumour regression. Here we use a spontaneously self-regressing tumour, cutaneous keratoacanthoma (KAs), to identify physiological mechanisms that drive tumour regression. By using a mouse model system that recapitulates the behaviour of human KAs, we show that self-regressing tumours shift their balance to a differentiation programme during regression. Furthermore, we demonstrate that developmental programs utilized for skin hair follicle regeneration, such as Wnt, are hijacked to sustain tumour growth and that the retinoic acid (RA) signalling pathway promotes tumour regression by inhibiting Wnt signalling. Finally, we find that RA signalling can induce regression of malignant tumours that do not normally spontaneously regress, such as squamous cell carcinomas. These findings provide new insights into the physiological mechanisms of tumour regression and suggest therapeutic strategies to induce tumour regression.

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RA induces SCC tumour regression.(a) RNA levels of RA in mouse SCC, KA in growth and regression. Data are represented as mean±s.d. (n=5 for each tumour analysed, *<0.05 obtained by unpaired t-test statistical analysis). (b) Immunofluorescence staining for Crabp2 in human SCC tumours, growing and regressing KAs (n=2 for each tumour). (c, left and central panel) Mouse treated with RA showing skin tumour regression (including SCC in the micrographs). (right panel) % of tumour growth rate by comparing untreated, mock-treated and RA-treated skin tumours at the fourth week post RA treatment. Red boxes indicate SCC tumours that have been diagnosed for each category, black box indicate KA and papilloma tumours (****<0.001 obtained by unpaired t-test statistical analysis). (d) qRT–PCR analysis for proliferation and differentiation genes in mock- and RA-injected SCC tumours. RA- versus mock-injected tumour comparison has been conducted by using the ΔΔCT method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript. Data are represented as mean±s.d. (n=7). (e) qRT–PCR analysis for Wnt target genes in mock- and RA-injected SCC tumours. RA versus mock-injected tumours comparison has been conducted by using the ΔΔCT method and normalized to GAPDH transcript. Data are represented as mean±s.d. (n=5). (f) Immunofluorescence staining for Sox9 in mock- and RA-injected SCC tumours (scale bar, 50 μm).
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f7: RA induces SCC tumour regression.(a) RNA levels of RA in mouse SCC, KA in growth and regression. Data are represented as mean±s.d. (n=5 for each tumour analysed, *<0.05 obtained by unpaired t-test statistical analysis). (b) Immunofluorescence staining for Crabp2 in human SCC tumours, growing and regressing KAs (n=2 for each tumour). (c, left and central panel) Mouse treated with RA showing skin tumour regression (including SCC in the micrographs). (right panel) % of tumour growth rate by comparing untreated, mock-treated and RA-treated skin tumours at the fourth week post RA treatment. Red boxes indicate SCC tumours that have been diagnosed for each category, black box indicate KA and papilloma tumours (****<0.001 obtained by unpaired t-test statistical analysis). (d) qRT–PCR analysis for proliferation and differentiation genes in mock- and RA-injected SCC tumours. RA- versus mock-injected tumour comparison has been conducted by using the ΔΔCT method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript. Data are represented as mean±s.d. (n=7). (e) qRT–PCR analysis for Wnt target genes in mock- and RA-injected SCC tumours. RA versus mock-injected tumours comparison has been conducted by using the ΔΔCT method and normalized to GAPDH transcript. Data are represented as mean±s.d. (n=5). (f) Immunofluorescence staining for Sox9 in mock- and RA-injected SCC tumours (scale bar, 50 μm).

Mentions: If RA/Wnt signalling is able to induce physiological tumour regression, we reasoned that the same cross-talk could induce the regression of skin tumours that do not spontaneously regress. To address this question, we used skin SCC as a model system. SCC tumours start to develop on the mouse back skin after 20 weeks of DMBA treatment. We first compared the levels of RA machinery in growing KAs, regressing KAs and SCCs. We found that RA signalling levels were lower in SCC tumours in comparison with regressing KA, as shown by qRT–PCR analysis for RA machinery (Fig. 7a). Consistent with these findings, Wnt ligands and inhibitors level in SCCs were comparable with KA in growth (Supplementary Fig. 8a). To test if RA dynamic regulation occurs in human skin tumour, we stained SCC tumours, as well as growing and regressing KAs for the RA-associated protein Crabp2. Crabp2 translocates from the cytoplasm to the nucleus to activate RA pathway. Strikingly, we found that Crabp2 was nuclear in regressing human KAs while it was predominantly cytoplasmic in growing KA and SCC human tumours (Fig. 7b). These findings prompted us to ask whether forced expression of RA signalling could result in regression of SCC tumours (Supplementary Fig. 8b). To address this question, we have injected RA or mock to SCCs for 7 consecutive days and waited for 4 weeks. RA activation was confirmed by qRT–PCR analysis post RA treatment and by nuclear staining for Crabp2 (Supplementary Fig. 8b,c). At 4-week post treatment, we found that RA injection led to regression of both KAs/papillomas (black) and SCCs (red) as quantified over time by macroscopic measurements (Fig. 7c). qRT–PCR analysis of RA and mock-injected SCC tumours showed that tumour regression was driven by loss of proliferation and increased differentiation programs (Fig. 7d). In addition, qRT–PCR analysis of Wnt target genes, as well as immunohistochemistry for Sox9 and β-catenin, showed that RA treatment induced Wnt inhibition in regressing SCCs (Fig. 7e,f and Supplementary Fig. 8d).


Spontaneous tumour regression in keratoacanthomas is driven by Wnt/retinoic acid signalling cross-talk.

Zito G, Saotome I, Liu Z, Ferro EG, Sun TY, Nguyen DX, Bilguvar K, Ko CJ, Greco V - Nat Commun (2014)

RA induces SCC tumour regression.(a) RNA levels of RA in mouse SCC, KA in growth and regression. Data are represented as mean±s.d. (n=5 for each tumour analysed, *<0.05 obtained by unpaired t-test statistical analysis). (b) Immunofluorescence staining for Crabp2 in human SCC tumours, growing and regressing KAs (n=2 for each tumour). (c, left and central panel) Mouse treated with RA showing skin tumour regression (including SCC in the micrographs). (right panel) % of tumour growth rate by comparing untreated, mock-treated and RA-treated skin tumours at the fourth week post RA treatment. Red boxes indicate SCC tumours that have been diagnosed for each category, black box indicate KA and papilloma tumours (****<0.001 obtained by unpaired t-test statistical analysis). (d) qRT–PCR analysis for proliferation and differentiation genes in mock- and RA-injected SCC tumours. RA- versus mock-injected tumour comparison has been conducted by using the ΔΔCT method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript. Data are represented as mean±s.d. (n=7). (e) qRT–PCR analysis for Wnt target genes in mock- and RA-injected SCC tumours. RA versus mock-injected tumours comparison has been conducted by using the ΔΔCT method and normalized to GAPDH transcript. Data are represented as mean±s.d. (n=5). (f) Immunofluorescence staining for Sox9 in mock- and RA-injected SCC tumours (scale bar, 50 μm).
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f7: RA induces SCC tumour regression.(a) RNA levels of RA in mouse SCC, KA in growth and regression. Data are represented as mean±s.d. (n=5 for each tumour analysed, *<0.05 obtained by unpaired t-test statistical analysis). (b) Immunofluorescence staining for Crabp2 in human SCC tumours, growing and regressing KAs (n=2 for each tumour). (c, left and central panel) Mouse treated with RA showing skin tumour regression (including SCC in the micrographs). (right panel) % of tumour growth rate by comparing untreated, mock-treated and RA-treated skin tumours at the fourth week post RA treatment. Red boxes indicate SCC tumours that have been diagnosed for each category, black box indicate KA and papilloma tumours (****<0.001 obtained by unpaired t-test statistical analysis). (d) qRT–PCR analysis for proliferation and differentiation genes in mock- and RA-injected SCC tumours. RA- versus mock-injected tumour comparison has been conducted by using the ΔΔCT method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript. Data are represented as mean±s.d. (n=7). (e) qRT–PCR analysis for Wnt target genes in mock- and RA-injected SCC tumours. RA versus mock-injected tumours comparison has been conducted by using the ΔΔCT method and normalized to GAPDH transcript. Data are represented as mean±s.d. (n=5). (f) Immunofluorescence staining for Sox9 in mock- and RA-injected SCC tumours (scale bar, 50 μm).
Mentions: If RA/Wnt signalling is able to induce physiological tumour regression, we reasoned that the same cross-talk could induce the regression of skin tumours that do not spontaneously regress. To address this question, we used skin SCC as a model system. SCC tumours start to develop on the mouse back skin after 20 weeks of DMBA treatment. We first compared the levels of RA machinery in growing KAs, regressing KAs and SCCs. We found that RA signalling levels were lower in SCC tumours in comparison with regressing KA, as shown by qRT–PCR analysis for RA machinery (Fig. 7a). Consistent with these findings, Wnt ligands and inhibitors level in SCCs were comparable with KA in growth (Supplementary Fig. 8a). To test if RA dynamic regulation occurs in human skin tumour, we stained SCC tumours, as well as growing and regressing KAs for the RA-associated protein Crabp2. Crabp2 translocates from the cytoplasm to the nucleus to activate RA pathway. Strikingly, we found that Crabp2 was nuclear in regressing human KAs while it was predominantly cytoplasmic in growing KA and SCC human tumours (Fig. 7b). These findings prompted us to ask whether forced expression of RA signalling could result in regression of SCC tumours (Supplementary Fig. 8b). To address this question, we have injected RA or mock to SCCs for 7 consecutive days and waited for 4 weeks. RA activation was confirmed by qRT–PCR analysis post RA treatment and by nuclear staining for Crabp2 (Supplementary Fig. 8b,c). At 4-week post treatment, we found that RA injection led to regression of both KAs/papillomas (black) and SCCs (red) as quantified over time by macroscopic measurements (Fig. 7c). qRT–PCR analysis of RA and mock-injected SCC tumours showed that tumour regression was driven by loss of proliferation and increased differentiation programs (Fig. 7d). In addition, qRT–PCR analysis of Wnt target genes, as well as immunohistochemistry for Sox9 and β-catenin, showed that RA treatment induced Wnt inhibition in regressing SCCs (Fig. 7e,f and Supplementary Fig. 8d).

Bottom Line: A fundamental goal in cancer biology is to identify the cells and signalling pathways that are keys to induce tumour regression.Furthermore, we demonstrate that developmental programs utilized for skin hair follicle regeneration, such as Wnt, are hijacked to sustain tumour growth and that the retinoic acid (RA) signalling pathway promotes tumour regression by inhibiting Wnt signalling.Finally, we find that RA signalling can induce regression of malignant tumours that do not normally spontaneously regress, such as squamous cell carcinomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Yale Stem Cell Center, Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut 06510, USA.

ABSTRACT
A fundamental goal in cancer biology is to identify the cells and signalling pathways that are keys to induce tumour regression. Here we use a spontaneously self-regressing tumour, cutaneous keratoacanthoma (KAs), to identify physiological mechanisms that drive tumour regression. By using a mouse model system that recapitulates the behaviour of human KAs, we show that self-regressing tumours shift their balance to a differentiation programme during regression. Furthermore, we demonstrate that developmental programs utilized for skin hair follicle regeneration, such as Wnt, are hijacked to sustain tumour growth and that the retinoic acid (RA) signalling pathway promotes tumour regression by inhibiting Wnt signalling. Finally, we find that RA signalling can induce regression of malignant tumours that do not normally spontaneously regress, such as squamous cell carcinomas. These findings provide new insights into the physiological mechanisms of tumour regression and suggest therapeutic strategies to induce tumour regression.

Show MeSH
Related in: MedlinePlus