Analysis of the Role of Igf2 in Adrenal Tumour Development in Transgenic Mouse Models

International audienceAdrenal cortical carcinomas (ACC) are rare but aggressive tumours associated with poor prognosis. The two most frequent alterations in ACC in patients are overexpression of the growth factor IGF2 and constitutive activation of Wnt/β-catenin signalling. Using a transgenic mouse model, we have previously shown that constitutive active β-catenin is a bona fide adrenal oncogene. However, although all these mice developed benign adrenal hyperplasia, malignant progression was infrequent, suggesting that secondary genetic events were required for aggressive tumour development. In the present paper, we have tested IGF2 oncogenic properties by developing two distinct transgenic mouse models of Igf2 overexpression in the adrenal cortex. Our analysis shows that despite overexpression levels ranging from 7 (basal) to 87 (ACTH-induced) fold, Igf2 has no tumour initiating potential in the adrenal cortex. However, it induces aberrant accumulation of Gli1 and Pod1-positive progenitor cells, in a hedgehog-independent manner. We have also tested the hypothesis that Igf2 may cooperate with Wnt signalling by mating Igf2 overexpressing lines with mice that express constitutive active β-catenin in the adrenal cortex. We show that the combination of both alterations has no effect on tumour phenotype at stages when β-catenin-induced tumours are benign. However, there is a mild promoting effect at later stages, characterised by increased Weiss score and proliferation. Formation of malignant tumours is nonetheless a rare event, even when Igf2 expression is further increased by ACTH treatment. Altogether these experiments suggest that the growth factor IGF2 is a mild contributor to malignant adrenocortical tumourigenesis

mTOR pathway is activated by PKA in adrenocortical cells and participates in vivo to apoptosis resistance in primary pigmented nodular adrenocortical disease (PPNAD).

International audience: Primary pigmented nodular adrenocortical disease (PPNAD) is associated with inactivating mutations of the PRKAR1A tumor suppressor gene that encodes the regulatory subunit R1α of the cAMP-dependent protein kinase (PKA). In human and mouse adrenocortical cells, these mutations lead to increased PKA activity, which results in increased resistance to apoptosis that contributes to the tumorigenic process. We used in vitro and in vivo models to investigate the possibility of a crosstalk between PKA and mammalian target of rapamycin (mTOR) pathways in adrenocortical cells and its possible involvement in apoptosis resistance. Impact of PKA signaling on activation of the mTOR pathway and apoptosis was measured in a mouse model of PPNAD (AdKO mice), in human and mouse adrenocortical cell lines in response to pharmacological inhibitors and in PPNAD tissues by immunohistochemistry. AdKO mice showed increased mTOR complex 1 (mTORC1) pathway activity. Inhibition of mTORC1 by rapamycin restored sensitivity of adrenocortical cells to apoptosis in AdKO but not in wild-type mice. In both cell lines and mouse adrenals, rapid phosphorylation of mTORC1 targets including BAD proapoptotic protein was observed in response to PKA activation. Accordingly, BAD hyperphosphorylation, which inhibits its proapoptotic activity, was increased in both AdKO mouse adrenals and human PPNAD tissues. In conclusion, mTORC1 pathway is activated by PKA signaling in human and mouse adrenocortical cells, leading to increased cell survival, which is correlated with BAD hyperphosphorylation. These alterations could be causative of tumor formation

Igf2 overexpression does not initiate adrenal tumourigenesis. A- β-catenin is not activated in AdIgf2 adrenals.

<p>β-catenin expression was analysed by immunohistochemistry in wild-type (WT, a), ΔCat (positive control, b) and AdIgf2 (c–d) adrenals. Section in c was counterstained with haemotxylin. Nucleo-cytoplasmic staining of β-catenin (white arrowheads) is restricted to zona glomerulosa in WT and AdIgf2 adrenals but spreads throughout the cortex and inside the medulla in ΔCat adrenals. Black arrows show infiltrating mesenchymal subcapsular cells. <b>B -Wnt pathway is not activated in AdIgf2 adrenals.</b> Expression levels of Axin2 and Lef-1, two canonical Wnt pathway targets, were determined by RTqPCR with cDNAs from wild-type, AdIgf2 and ΔCat (positive control) adrenals. Bars represent the mean relative quantification (Rq Tg/WT) of gene expression for each gene in at least 6 adrenals per genotype ± standard deviation. <i>P</i>-value was calculated using Student's <i>t</i>-test. <b>C- Analysis of Ki67 expression.</b> Ki67 was detected by immunohistochemistry on wild-type (WT, a) and AdIgf2 adrenals (b). <b>D- </b><b>Proliferation is not increased in AdIgf2 adrenals.</b> Numbers of Ki67-positive cells in a whole adrenal section were counted separately in the cortex (Co) and medulla (M). For each zone, the number of cells was corrected for surface and is expressed as a percentage of Ki67-positive cells in one of the control individuals. Bars represent the mean of at least 7 individual counts in wild-type and AdIgf2 adrenals ± standard deviation. <i>P</i>-value was calculated using Student's <i>t</i>-test. <b>E- Cyclin D1 expression is not increased in AdIgf2 adrenals.</b> Expression of Cyclin D1 was analysed by RTqPCR with cDNAs from Wild-type and AdIgf2 adrenals. Bars represent the mean relative quantification (Rq AdIgf2/WT) of gene expression in at least 5 adrenals per genotype ± standard deviation. <i>P</i>-value was calculated using Student's <i>t</i>-test. <b>F- Igf2 overexpression does not induce adrenal tumour formation.</b> The phenotype of AdIgf2 adrenals was followed over a 14 months time course by haematoxylin & eosin staining (b–c) and immunohistochemistry for Ki67 (e–f). A 10 month-old wild-type adrenal was included as a reference (WT, a, d). Some twelve month-old AdIgf2 mice were treated with ACTH for two months in order to increase Igf2 expression. Adrenal histology and proliferation were then assessed by haematoxylin & eosin staining (g) and Ki67 immunohistochemistry (h). Arrows show mesenchymal subcapsular cells. M, medulla; F, fasciculata; G, glomerulosa; Co, cortex. Scale bar is 80 µm.</p

Characterisation of AdIgf2 transgenic mice adrenals. A- Igf2 is overexpressed in the adrenals of transgenic mice.

<p>Igf2 expression was analysed by RTqPCR on cDNAs from adrenals, kidneys, liver, spleen, ovaries and testes of 10 month-old wild-type (WT) and AdIgf2 transgenic mice. Bars represent the mean relative quantification (Rq AdIgf2/WT) of Igf2 expression for each tissue in at least 4 samples per genotype ± standard deviation. <i>P</i>-value was calculated using Student's <i>t</i>-test. <b>B- Igf2 signalling is increased in AdIgf2 adrenals.</b> Expression of Igf2 (a–b) and of the phosphorylated ribosomal protein S6 (c–d) was analysed by immunohistochemistry in WT and AdIgf2 adrenals. <b>C- Effect of Igf2 overexpression on adrenal histology and differentiation.</b> Histology was analysed by haematoxylin & eosin staining in wild-type (a) and AdIgf2 (b) adrenals. Expression of Sf-1 (c–d), Akr1b7 (e–f) and Dab2 (g–h) was analysed by immunohistochemistry in WT and AdIgf2 adrenals. Black arrows show infiltrating mesenchymal subcapsular cells. <b>D- Igf2 and phospho-S6 are not expressed in subcapuslar mesenchymal cells.</b> Igf2 (a) and phospho-S6 (b) were immunodetected in AdIgf2 adrenals and the tissue was counterstained with haematoxylin. <b>E- Adrenal progenitor cells markers are overexpressed in AdIgf2 adrenal.</b> Expression of the progenitor cells markers, Gli1, Ptch1, Pod1 and Shh was analysed by RTqPCR on cDNAs from wild-type (WT) and AdIgf2 adrenals. Bars represent the mean relative quantification (Rq AdIgf2/WT) of gene expression for each marker in at least 6 adrenals per genotype ± standard deviation. <i>P</i>-value was calculated using Student's <i>t</i>-test. <b>F- Pod1 and Gli1 are overexpressed in AdIgf2 adrenals.</b> Pod1 and Gli1 expression domains were analysed by in-situ hybridisation in wild-type (a, c) and AdIgf2 (b, d) adrenals. In all panels, arrows show mesenchymal subcapsular cells. M, medulla; F, fasciculata; G, glomerulosa; Co, cortex; Ca, capsule. Scale bar is 80 µm.</p

Analysis of the cooperation between β-catenin activation and Igf2 overexpression in 10 month-old mice. A- Igf2 overexpression in 10 month-old ΔCat mice does not significantly alter the adrenal phenotype.

<p>AdIgf2 mice were mated with ΔCat mice to generate ΔCat;AdIgf2 compound animals. The adrenal phenotype in 10 month-old mice was analysed by haematoxylin & eosin staining (a–c) and immunohistochemistry for Dab2. Black arrows show infiltrating subcapsular mesenchymal cells. <b>B- Analysis of Ki67 expression in 10 month-old ΔCat;AdIgf2 adrenals.</b> Ki67 expression was analysed by immunohistochemistry in wild-type (B, a), ΔCat (B, b) and ΔCat;Igf2 (B, c) adrenals. <b>C- Igf2 overexpression in ΔCat mice does not significantly increase proliferation. a-Counting of Ki67-positive cells.</b> Numbers of Ki67-positive cells in a whole adrenal section were counted separately in the cortex (Co) and medulla (M). For each zone, the number of cells was corrected for surface and is expressed as a percentage of Ki67-positive cells in one of the control individuals. Bars represent the mean of at least 7 individual counts in wild-type, ΔCat and ΔCat;AdIgf2 adrenals ± standard deviation. <i>P</i>-value was calculated using Student's <i>t</i>-test. <b>b- Analysis of Cyclin D1 expression.</b> Cyclin D1 expression was analysed by RTqPCR with cDNAs from wild-type, ΔCat and ΔCat;AdIgf2 adrenals. Bars represent the mean relative quantification (Rq Tg/WT) of gene expression in at least 7 adrenals per genotype ± standard deviation. <i>P</i>-value was calculated using Student's <i>t</i>-test. <b>D- Analysis of malignancy markers expression.</b> Expression of VegfA, Connexina43 (cnx43) and Nov/Ccn3 was analysed by RTqPCR with cDNAs from wild-type (WT), ΔCat and ΔCat;AdIgf2 adrenals. Bars represent the mean relative quantification (Rq Tg/WT) of gene expression for each marker in at least 7 adrenals per genotype ± standard deviation. <i>P</i>-value was calculated using Student's <i>t</i>-test. M, medulla; F, fasciculata; G, glomerulosa. Scale bar is 80 µm.</p