Inactivation of TGF-β signaling and loss of PTEN cooperate to induce colon cancer in vivo

Abstract

The accumulation of genetic and epigenetic alterations mediates colorectal cancer (CRC) formation by deregulating key signaling pathways in cancer cells. In CRC, one of the most commonly inactivated signaling pathways is the transforming growth factor-beta (TGF-β) signaling pathway, which is often inactivated by mutations of TGF-β type II receptor (TGFBR2). Another commonly deregulated pathway in CRC is the phosphoinositide-3-kinase (PI3K)-AKT pathway. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of PI3K-AKT signaling and is silenced in ∼30% of CRC. The combination of TGFBR2 inactivation and loss of PTEN is particularly common in microsatellite-unstable CRCs. Consequently, we determined in vivo if deregulation of these two pathways cooperates to affect CRC formation by analyzing tumors arising in mice that lack Tgfbr2 and/or Pten specifically in the intestinal epithelium. We found that lack of Tgfbr2 (Tgfbr2(IEKO)) alone is not sufficient for intestinal tumor formation and lack of Pten (Pten(IEKO)) alone had a weak effect on intestinal tumor induction. However, the combination of Tgfbr2 inactivation with Pten loss (Pten(IEKO);Tgfbr2(IEKO)) led to malignant tumors in both the small intestine and colon in 86% of the mice and to metastases in 8% of the tumor-bearing mice. Moreover, these tumors arose via a β-catenin-independent mechanism. Inactivation of TGF-β signaling and loss of Pten in the tumors led to increased cell proliferation, decreased apoptosis and decreased expression of cyclin-dependent kinase inhibitors. Thus, inactivation of TGF-β signaling and loss of PTEN cooperate to drive intestinal cancer formation and progression by suppressing cell cycle inhibitors.

Figure 1. Inactivation of Tgf-β signaling and loss of Pten lead to markedly reduced survival

Kaplan-Meier analysis reveals a median survival of 36 weeks in Pten^IEKO;Tgfbr2^IEKO mice (N=43). A significant decrease in the survival of Pten^IEKO;Tgfbr2^IEKO mice was observed compared to Tgfbr2^IEKO mice, Pten^IEKO mice or Control mice (P < 0.0001, log-rank test, for all comparisons). There was no significant difference in survival when the Tgfbr2^IEKO mice or Pten^IEKO mice were compared to Control mice (P > 0.1 for both comparisons, log-rank test). Of note, all mice that survived to 54 weeks of age were sacrificed at that age.

Figure 2. Inactivation of Tgf-β signaling and loss of Pten lead to formation of invasive adenocarcinomas

(A) Low-power image of a representative colonic mucinous adenocarcinoma from a Pten^IEKO;Tgfbr2^IEKO mouse (H&E, original magnification: 20X). There is transmural effacement by neoplastic glands and large lakes of tumor-produced mucin. In some cases, mucin lakes extend through the serosa and into the serosal lymphatics. The inset was taken from the boxed region in (A) at higher magnification and the arrow points to intravascular neoplastic cells within the primary colonic mucinous adenocarcinoma (H&E, original magnification: 200X). (B) Low-power image of a liver section reveals intravascular neoplastic cells (H&E, original magnification: 100X). The inset was taken from the boxed region in (B) at higher magnification and the arrow points to the intravascular neoplastic cells (H&E, original magnification, 200X). (C) Low-power image of a mesenteric metastasis arising from a Pten^IEKO;Tgfbr2^IEKO mouse (H&E, original magnification: 40X). The inset was taken from the boxed region in (C) at higher magnification to show the neoplastic glands and mucin production (H&E, original magnification: 200X). (D) Low-power image of Alcian blue stained section of the same mesenteric metastasis shown in panel C (Alcian blue stain, original magnification: 40X). The inset was taken from the boxed region in (D) at higher magnification to show the mucin production in this metastasis (Alcian blue stain, original magnification: 200X). (E) E-cadherin immunostaining of the same mesentery metastasis shown in (C) (E-cadherin IHC, original magnification: 40X). The inset was taken from the boxed region in (E) at higher magnification to demonstrate strong expression of E-cadherin, consistent with the epithelial origin of this metastasis (original magnification: 200X). (F) PCR genotyping results demonstrate the recombination of the Tgfbr2^flx allele in peritoneal metastasis (PT met1 and PT met2) and mesentery metastases (MSE met1 and MSE met2). The positive control DNA is from the small intestine mucosa of a Tgfbr2^IEKO mouse, and the negative control DNA is from the lymph nodes of a Pten^IEKO;Tgfbr2^IEKO mouse that did not have grossly evident metastatic disease.

Figure 3. Signaling pathway deregulation in Pten^IEKO;Tgfbr2^IEKO tumors

(A) Low-power image of a representative intestinal adenocarcinoma from a Pten^IEKO;Tgfbr2^IEKO mouse (H&E, original magnification: 20X). The boxed region in (A) is shown at higher magnification in the adjacent photomicrographs of immunostained tissue. Neoplastic epithelial cells in Pten^IEKO;Tgfbr2^IEKO mice show (A) decreased PTEN expression by PTEN immunostaining and (B) increased Akt activation by phospho-Akt (Ser473) immunostaining. The invasive front of a representative adenocarcinoma shows (C) membrane-bound β-catenin. (A–C, original magnification 200X). (D) Upper level, analysis of activation of Akt by immunoblotting of lysates from normal mucosa of wild-type (WT) control and Tgfbr2^IEKO mice (left panel); in normal mucosa of Pten^IEKO mice, normal mucosa and tumors from Pten^IEKO;Tgfbr2^IEKO mice (right panel). β-actin was used as a loading control. Middle level and lower level, relative Akt phosphorylation levels at Thr308 and Ser473 sites as determined by densitometry, normalized to total Akt and further normalized to β-actin. Note a significant decrease in relative Akt phosphorylation levels at Thr308 and Ser473 sites in tumors tissue (T) compared to normal tissue (N) from Pten^IEKO;Tgfbr2^IEKO mice. (* p<0.05 for both relative pAkt T308 and S473, Mann-Whitney test) (E) Upper level, analysis of activation of ERK1/2 by immunoblotting of lysates from normal mucosa of wild-type Control and Tgfbr2^IEKO mice (left panel); in normal mucosa of Pten^IEKO mice, normal mucosa and tumors from Pten^IEKO;Tgfbr2^IEKO mice (right panel). β-actin was used as a loading control. Lower level, relative ERK1/2 phosphorylation levels as determined by densitometry, normalized to total ERK1/2 and further normalized to β-actin. Note a significant increase in relative ERK1/2 phosphorylation levels in tumor tissue (T) compared to normal tissue (N) from Pten^IEKO;Tgfbr2^IEKO mice. (* p<0.05, Mann-Whitney test).

Figure 4. Assessment of proliferation, apoptosis and cyclin dependent kinase inhibitors in normal tissue and Pten^IEKO;Tgfbr2^IEKO tumors

(A) Representative photomicrograph of Ki67 immunostained tumor from Pten^IEKO;Tgfbr2^IEKO mice. (B) Representative photomicrograph of cleaved caspase-3 immunostained tumor from Pten^IEKO;Tgfbr2^IEKO mice. Rare stained cells are present and stained cellular debris is noted within the lumen of neoplastic glands (arrow). (C) Assessment of p15^Ink4b (Cdkn2b), p21^Cip1 (Cdkn1a) and p27^Kip1(Cdkn1b) mRNA by qRT-PCR in matched normal mucosa and tumors from Pten^IEKO;Tgfbr2^IEKO mice. (Mann-Whitney test, * p=0.01 for p15^Ink4b(Cdkn2b), 0.0033 for p21^Cip1(Cdkn1a), and 0.015 for p27^Kip1(Cdkn1b)) (D) Assessment of p15^Ink4b, p21^Cip1 and p27^Kip1 protein levels by immunoblotting lysates from normal mucosa of various genotypes, and normal mucosa and tumors from the Pten^IEKO;Tgfbr2^IEKO mice. GAPDH was used as a loading control. Normalized protein levels of p15^Ink4b, p21^Cip1 and p27^Kip1 were determined by densitometry, normalized to GAPDH. Note a significant decrease in normalized levels of p15^Ink4b, p21^Cip1 and p27^Kip1 in tumor tissue (T) compared to normal tissue (N) from Pten^IEKO;Tgfbr2^IEKO mice. (* p<0.05, Mann-Whitney test, N=3)

Figure 5. Reconstitution of TGFBR2 and PTEN induce p21^CIP1 expression in colon cancer cells

Stimulation of p21Pδ2.1 reporter luciferase activity in the colon cancer cell line SNU-C4 transfected with empty vector (EV), TGFBR2, PTEN, or both TGFBR2 and PTEN. Transfected SNU-C4 cells were treated with TGF-β (2 ng/ml). Histograms represent relative luciferase activity normalized to Renilla luciferase reporter activity. The error bars represent the standard error from three independent experiments. The 3TP-lux luciferase reporter was used to confirm the specificity of the cooperation between TGFBR2 and PTEN on the activation of p21^CIP1 luciferase reporter. Reconstitution of the SNU-C4 cells with PTEN alone did not induce 3TP-lux activity. Furthermore, there was no increase in the luciferase activity of the 3TP-lux reporter in TGFBR2 and PTEN co-transfected cells compared to TGFBR2-transfected alone cells, indicating the specificity of the additive effect of PTEN and TGFBR2 on p21^CIP1 expression.