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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-ofprinciple, 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. EMBO reports 4, 609-615 (2003) doi:10.1038/sj.embor.embor865 INTRODUCTIONThe transactivation of T-cell factor (TCF) target genes induced by wingless-related (WNT) pathway mutations is the main transforming event in colorectal cancer (CRC; Kinzler & Vogelstein, 1996;Bienz & Clevers, 2000). We have recently studied the TCF target-gene programme by the inducible overexpression of dominant-negative versions of TCF1 and TCF4 in CRC cell lines (van de Wetering et al., 2002;Batlle et al., 2002). This overexpression disrupted endogenous β-catenin/TCF4 activity in CRC cells. Importantly, it induced rapid G1 arrest and blocked a genetic programme that is physiologically active in the proliferative compartment of colon crypts. Consequently, an intestinal differentiation programme was induced. We concluded that the β-catenin/TCF4 complex is the master switch that controls the decision between proliferation versus differentiation in healthy and malignant intestinal epithelial cells.As the overexpression of dominant-negative proteins might induce artefactual effects, we used a loss-of-function strategy to confirm our results. It is possible to carry out classical gene knockouts by homologous recombination in CRC cells (Shirasawa et al., 1993;Chan et al., 1999Chan et al., , 2002Kim et al., 2002;Sekine et al., 2002), but this technology is relatively time-consuming. Moreover, as we expected a growth-arrest phenotype, clones would either fail to develop or would be selected for other growth-promoting events. More recently, RNA interference (RNAi), a well-established method for gene knockdown in model organisms (Sharp, 2001), can also be used for gene knockdown in mammalian cells (Elbashir et al. 2001). So-called small interfering RNAs (siRNAs) have been introduced into mammalian cells by the transient transfection of synthetic doublestranded RNA. Alternatively, promoters of genes transcribed by RNA polymerase III have been used to drive the expression of hairpin RNAs, which are very similar to siRNAs (Brummelkamp et al., 2002;Miyagishi & Taira, 2002;Paul et al., 200...

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.

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.

SUMMARY 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.