Vanesa Muncan

Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector

We have designed a doxycycline‐regulated form of the H1 promoter of RNA polymerase III that allows the inducible knockdown of gene expression by small interfering RNAs (siRNAs). As a proof‐of‐principle, we have targeted β‐catenin in colorectal cancer (CRC) cells. T‐cell factor (TCF) target‐gene expression is induced by accumulated β‐catenin, and is the main transforming event in these cells. We have shown previously that the disruption of β‐catenin/TCF4 activity in CRC cells by the overexpression of dominant‐negative TCF induces rapid G1 arrest and differentiation. Stable integration of our inducible siRNA vector allowed the rapid production of siRNAs on doxycycline induction, followed by specific downregulation of β‐catenin. In these CRC cells, TCF reporter‐gene activity was inhibited, and G1 arrest and differentiation occurred. The inhibition of two other genes using this vector system shows that it should be useful for the inducible knockdown of gene expression.

Myc deletion rescues Apc deficiency in the small intestine

The APC gene encodes the adenomatous polyposis coli tumour suppressor protein, germline mutation of which characterizes familial adenomatous polyposis (FAP), an autosomal intestinal cancer syndrome. Inactivation of APC is also recognized as the key early event in the development of sporadic colorectal cancers, and its loss results in constitutive activity of the β-catenin–Tcf4 transcription complex. The proto-oncogene c-MYC has been identified as a target of the Wnt pathway in colorectal cancer cells in vitro, in normal crypts in vivo and in intestinal epithelial cells acutely transformed on in vivo deletion of the APC gene; however, the significance of this is unclear. Therefore, to elucidate the role Myc has in the intestine after Apc loss, we have simultaneously deleted both Apc and Myc in the adult murine small intestine. Here we show that loss of Myc rescued the phenotypes of perturbed differentiation, migration, proliferation and apoptosis, which occur on deletion of Apc. Remarkably, this rescue occurred in the presence of high levels of nuclear β-catenin. Array analysis revealed that Myc is required for the majority of Wnt target gene activation following Apc loss. These data establish Myc as the critical mediator of the early stages of neoplasia following Apc loss.

Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc.

ABSTRACT Inhibition of the mutationally activated Wnt cascade in colorectal cancer cell lines induces a rapid G1 arrest and subsequent differentiation. This arrest can be overcome by maintaining expression of a single Tcf4 target gene, the proto-oncogene c-Myc. Since colorectal cancer cells share many molecular characteristics with proliferative crypt progenitors, we have assessed the physiological role of c-Myc in adult crypts by conditional gene deletion. c-Myc-deficient crypts are lost within weeks and replaced by c-Myc-proficient crypts through a fission process of crypts that have escaped gene deletion. Although c-Myc−/− crypt cells remain in the cell cycle, they are on average much smaller than wild-type cells, cycle slower, and divide at a smaller cell size. c-Myc appears essential for crypt progenitor cells to provide the necessary biosynthetic capacity to successfully progress through the cell cycle.

Focal Adhesion Kinase Is Required for Intestinal Regeneration and Tumorigenesis Downstream of Wnt/c-Myc Signaling

The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, we investigated the functional importance of FAK following deletion of the Apc tumor suppressor protein within the intestinal epithelium. Following Apc loss, FAK expression increased in a c-Myc-dependent manner. Codeletion of Apc and Fak strongly reduced proliferation normally induced following Apc loss, and this was associated with reduced levels of phospho-Akt and suppression of intestinal tumorigenesis in Apc heterozygous mice. Thus, FAK is required downstream of Wnt Signaling, for Akt/mTOR activation, intestinal regeneration, and tumorigenesis. Importantly, this work suggests that FAK inhibitors may suppress tumorigenesis in patients at high risk of developing colorectal cancer.

ER Stress Causes Rapid Loss of Intestinal Epithelial Stemness through Activation of the Unfolded Protein Response

Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER) stress and activity of the unfolded protein response (UPR) are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2α-dependent manner. Inhibition of Perk-eIF2α signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation.

Blimp1 regulates the transition of neonatal to adult intestinal epithelium.

In many mammalian species, the intestinal epithelium undergoes major changes that allow a dietary transition from mothers milk to the adult diet at the end of the suckling period. These complex developmental changes are the result of a genetic programme intrinsic to the gut tube, but its regulators have not been identified. Here we show that transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1) is highly expressed in the developing and postnatal intestinal epithelium until the suckling to weaning transition. Intestine-specific deletion of Blimp1 results in growth retardation and excessive neonatal mortality. Mutant mice lack all of the typical epithelial features of the suckling period and are born with features of an adult-like intestine. We conclude that the suckling to weaning transition is regulated by a single transcriptional repressor that delays epithelial maturation.

B-catenin deficiency, but not Myc deletion, suppresses the immediate phenotypes of APC loss in the liver

Dysregulated Wnt signaling is seen in approximately 30% of hepatocellular carcinomas; thus, finding pathways downstream of the activation of Wnt signaling is key. Here, using cre-lox technology, we deleted the Apc gene in the adult mouse liver and observed a rapid increase in nuclear β-catenin and c-Myc, which is associated with an induction of proliferation that led to hepatomegaly within 4 days of gene deletion. To investigate the downstream pathways responsible for these phenotypes, we analyzed the impact of inactivating APC in the context of deficiency of the potentially key effectors β-catenin and c-Myc. β-catenin loss rescues both the proliferation and hepatomegaly phenotypes after APC loss. However, c-Myc deletion, which rescues the phenotypes of APC loss in the intestine, had no effect on the phenotypes of APC loss in the liver. The consequences of the deregulation of the Wnt pathway within the liver are therefore strikingly different from those observed within the intestine, with the vast majority of Wnt targets being β-catenin-dependent but c-Myc-independent in the liver.

Cyclin D2–Cyclin-Dependent Kinase 4/6 Is Required for Efficient Proliferation and Tumorigenesis following Apc Loss

Inactivation of the Apc gene is recognized as the key early event in the development of sporadic colorectal cancer (CRC), where its loss leads to constitutive activation of β-catenin/T-cell factor 4 signaling and hence transcription of Wnt target genes such as c-Myc. Our and other previous studies have shown that although cyclin D1 is required for adenoma formation, it is not immediately upregulated following Apc loss within the intestine, suggesting that proliferation following acute Apc loss may be dependent on another D-type cyclin. In this study, we investigated the expression and functional relevance of cyclin D2 following Apc loss in the intestinal epithelium. Cyclin D2 is upregulated immediately following Apc loss, which corresponded with a significant increase in cyclin-dependent kinase 4 (CDK4) and hyperphosphorylated Rb levels. Deficiency of cyclin D2 resulted in a reduction in enterocyte proliferation and crypt size within Apc-deficient intestinal epithelium. Moreover, cyclin D2 dramatically reduced tumor growth and development in Apc(Min/+) mice. Importantly, cyclin D2 knockout did not affect proliferation of normal enterocytes, and furthermore, CDK4/6 inhibition also suppressed the proliferation of adenomatous cells and not normal cells from Apc(Min/+) mice. Taken together, these results indicate that cyclin D-CDK4/6 complexes are required for the efficient proliferation of cells with deregulated Wnt signaling, and inhibiting this complex may be an effective chemopreventative strategy in CRC.

T‐cell factor 4 (Tcf7l2) maintains proliferative compartments in zebrafish intestine

Previous studies have shown that Wnt signals, relayed through β‐catenin and T‐cell factor 4 (Tcf4), are essential for the induction and maintenance of crypts in mice. We have now generated a tcf4 (tcf7l2) mutant zebrafish by reverse genetics. We first observe a phenotypic defect at 4 weeks post‐fertilization (wpf), leading to death at about 6 wpf. The phenotype comprises a loss of proliferation at the base of the intestinal folds of the middle and distal parts of the intestine. The proximal intestine represents an independent compartment, as it expresses sox2 in the epithelium and barx1 in the surrounding mesenchyme, which are early stomach markers in higher vertebrates. Zebrafish are functionally stomach‐less, but the proximal intestine might share its ontogeny with the mammalian stomach. Rare adult homozygous tcf4−/− ‘escapers’ show proliferation defects in the gut epithelium, but have no other obvious abnormalities. This study underscores the involvement of Tcf4 in maintaining proliferative self‐renewal in the intestine throughout life.

Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo

Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.