3D imaging of colorectal cancer organoids identifies responses to Tankyrase inhibitors.

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.

Developmentally regulated Tcf7l2 splice variants mediate transcriptional repressor functions during eye formation.

Tcf7l2 mediates Wnt/ß-Catenin signalling during development and is implicated in cancer and type-2 diabetes. The mechanisms by which Tcf7l2 and Wnt/ß-Catenin signalling elicit such a diversity of biological outcomes are poorly understood. Here, we study the function of zebrafish tcf7l2alternative splice variants and show that only variants that include exon five or an analogous human tcf7l2 variant can effectively provide compensatory repressor function to restore eye formation in embryos lacking tcf7l1a/tcf7l1b function. Knockdown of exon five specific tcf7l2 variants in tcf7l1a mutants also compromises eye formation, and these variants can effectively repress Wnt pathway activity in reporter assays using Wnt target gene promoters. We show that the repressive activities of exon5-coded variants are likely explained by their interaction with Tle co-repressors. Furthermore, phosphorylated residues in Tcf7l2 coded exon5 facilitate repressor activity. Our studies suggest that developmentally regulated splicing of tcf7l2 can influence the transcriptional output of the Wnt pathway.

Transforming growth factor-beta1 mediates cellular response to DNA damage in situ.

Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

The potential for targeting oncogenic WNT/beta-catenin signaling in therapy.

There has been a surge of interest in the therapeutic targeting of the Wnt pathway following the demonstration that it is activated in a wide variety of tumors and that blocking aberrant signaling promoted tumor cell apoptosis or differentiation. This review describes recent therapeutic approaches and discusses potential opportunities for intervention at multiple levels within the Wnt pathway.

Deficiency of Mbd2 attenuates Wnt signaling.

We have previously shown that deficiency of the methyl binding domain protein Mbd2 dramatically reduces adenoma burden on an Apc(Min/+) background. To investigate the mechanism underlying this phenomenon, we have determined the effect of Mbd2 deficiency upon the phenotypes imposed by the conditional deletion of Apc in the small intestine. Microarray analysis demonstrated a partial suppression of the Wnt pathway in the absence of Mbd2. Mbd2 deficiency also influenced one immediate cellular consequence of Apc loss, with normalization of Paneth cell positioning. From a mechanistic perspective, we show that deficiency of Mbd2 elevates levels of the known Wnt target Lect2, and we confirm here that Mbd2 binds the Lect2 promoter in association with NuRD. Furthermore, we show that Lect2 is capable of functioning as a Wnt pathway repressor. These results therefore provide a mechanistic basis for the epigenetic control of adenoma formation mediated through Mbd2.

Proliferation of estrogen receptor-alpha-positive mammary epithelial cells is restrained by transforming growth factor-beta1 in adult mice.

Transforming growth factor (TGF)-beta1 is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor (ER)-alpha cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF-beta1 is necessary for the quiescence of ER-alpha-positive populations, we examined mouse mammary epithelial glands at estrus. Approximately 35% of epithelial cells showed TGF-beta1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF-beta signaling is autocrine. Nuclear Smad co-localized with nuclear ER-alpha. To test whether TGF-beta inhibits proliferation, we examined genetically engineered mice with different levels of TGF-beta1. ER-alpha co-localization with markers of proliferation (ie, Ki-67 or bromodeoxyuridine) at estrus was significantly increased in the mammary glands of Tgf beta1 C57/bl/129SV heterozygote mice. This relationship was maintained after pregnancy but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF-beta1 via the MMTV promoter suppressed proliferation of ER-alpha-positive cells. Thus, TGF-beta1 activation functionally restrains ER-alpha-positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF-beta1 dysregulation may promote proliferation of ER-alpha-positive cells associated with breast cancer risk in humans.

Latent transforming growth factor-beta activation in mammary gland: regulation by ovarian hormones affects ductal and alveolar proliferation.

Transforming growth factor-beta1 (TGF-beta 1) is a pluripotent cytokine that can inhibit epithelial proliferation and induce apoptosis, but is also widely implicated in breast cancer progression. Understanding its biological action in mammary development is critical for understanding its role in cancer. TGF-beta 1 is produced as a latent complex that requires extracellular activation before receptor binding. To better understand the spatial and temporal regulation of its action during mammary gland development, we examined the pattern of activation in situ using antibodies selected to distinguish between latent and active TGF-beta. Activation was highly restricted. TGF-beta 1 activation was localized primarily to the epithelium, and within the epithelium it was restricted to luminal epithelial cells but absent from either cap or myoepithelial cells. Within the luminal epithelium, we noted a further restriction. During periods of proliferation (ie, puberty, estrus and pregnancy), which are stimulated by ovarian hormones, TGF-beta 1 activation decreased in some cells, consistent with preparation for proliferation. Paradoxically, other cells simultaneously increase TGF-beta 1 immunoreactivity, which suggests that TGF-beta 1 differentially restrains epithelial subpopulations from responding to hormonal signals to proliferate. These data suggest that endogenous TGF-beta 1 activation and thus activity are regulated by ovarian hormones. To determine the specific consequences of TGF-beta 1 activity, we manipulated TGF-beta 1 levels in vivo using Tgfbeta 1 knockout mice and undertook tissue recombination experiments with heterozygous tissue. In Tgfbeta 1 heterozygous mice, which have <10% wild-type levels of TGF-beta1, ductal development during puberty and alveolar development during pregnancy were accelerated, consistent with its role as a growth inhibitor. The proliferative index of Tgfbeta 1+/- epithelium was increased approximately twofold in quiescent tissue and fourfold in proliferating tissue but both ducts and alveoli were grossly and histologically normal. To test whether epithelial TGF-beta1 was critical to the proliferative phenotype, Tgfbeta 1+/+ and +/- epithelium were transplanted into +/+ mammary stroma. The outgrowth of Tgfbeta 1+/- epithelium was accelerated in wild-type hosts, indicating that the phenotype was intrinsic to the epithelium. Moreover, proliferation was 15-fold greater in Tgfbeta 1+/- than wild-type mice after ovariectomy and treatment with estrogen and progesterone, suggesting that TGF-beta 1 acts in an autocrine or juxtacrine manner to regulate epithelial proliferation. Together these data indicate that ovarian hormones regulate TGF-beta 1 activation, which in turn restricts proliferative response to hormone signaling.