Bidirectional conversion of intestinal epithelial cells

Abstract

NFκB represents a key transcription factor within the inflammatory tumor microenvironment; however, NFκB’s function in tumor-initiating cells has not been examined yet. A recent Cell paper by Schwitalla et al. reports that NFκB modulates Wnt signaling and shows that enhanced Wnt activation induces dedifferentiation of nonstem cells that acquire tumor-initiating capacity.

Keywords: :

• EMT
• Lgr5
• NFκB
• Wnt signaling
• chronic inflammation
• dedifferentiation
• microenvironment

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In mammals, there are five members of the transcription factor NFκB family: RelA (p65), RelB, and c-Rel as well as the precursor proteins NFκB1 (p105) and NFκB2 (p100).¹ NFκB transcription factors bind to κB sites in promoters and enhancers of a variety of genes, inducing or repressing transcription. Bacterial and viral infection, inflammatory cytokines and engagement of antigen receptors all elicit NFκB activation, highlighting the crucial role of this transcription factor in the orchestration of immunity. It is thus not surprising that NFκB activity is tightly controlled at multiple levels by positive and negative regulatory elements.

Since its discovery 25 y ago, NFκB has served as a model system for inducible transcription and, because of its broad physiological and medical effects, has garnered tremendous research interest. Nevertheless, the diversity of NFκB function still raises questions about how a limited set of signaling mediators is able to integrate diverse stimuli to achieve a cell type- and stimulus-specific response. However, NFκB does not exist in isolation, and studies have begun to elucidate how crosstalk with parallel signaling networks shapes the NFκB response.

Inflammation is a manifestation of innate immunity, a fundamental protective response that is conserved in all multicellular animals.^2,3 Various forms of chronic inflammation increase the risk of several common cancers.⁴ Long-term administration of non-steroidal anti-inflammatory drugs (NSAIDs) can be tumor preventative and significantly reduce the incidence of many solid tumor entities, including colorectal cancer, suggesting a specific effect of low-grade inflammation in this context. However, the exact mechanism of chemoprevention by NSAIDs remains unknown. There is mounting evidence today that many tumors are propagated by means of cancer stem cells, rare cells in tumors with indefinite capacity for self-renewal.⁵ If that holds true, tissue stem cells should be closely guarded against infectious and chemical genotoxic insults and, at the same time, might be particularly vulnerable to deregulated innate immunity.

Although there is substantial evidence for a role of NFκB in tumor promotion and progression, so far its contribution to tumor initiation and epithelial tumor stem cell function has not been addressed. Here, Schwitalla et al. demonstrate that NFκB can enhance Wnt-signaling leading to dedifferentiation of epithelial non-stem cells into tumor initiating cells.

First, Schwitalla et al. showed that constitutive activation of β-catenin in intestinal epithelial cells (IEC) results in rapid expansion of intestinal crypt stem cells and TNFα-dependent NFκB activation. Second, Schwitalla et al. showed that inhibition of NFκB in IEC prolongs survival and delays crypt transformation. Together, these data suggest that NFκB could affect the development of a crypt progenitor phenotype and the initiation of adenomatous cell transformation through the regulation of Wnt-dependent intestinal stem cell gene expression.

Next, Schwitalla et al. enabled IEC-specific constitutive NFκB activity along with persistent β-catenin in vivo. Expectedly, animal survival was significantly shortened. Apart from a massive accumulation of highly proliferative crypt stem cells mice frequently displayed aberrant foci along the villus epithelium resembling crypt structures. These foci were actively proliferating and expressed stem cell markers suggesting that NFκB-mediated enhancement of β-catenin signaling in villus cells allowed a dedifferentiation program and occurrence of crypt stem cells in an aberrant position.

To confirm this hypothesis, Schwitalla et al. employed IEC from a recently developed organoid culture system based on the fact that oncogenic K-ras strongly cooperates with deregulated Wnt signaling.⁶ Villi isolated from K-ras compound mutants formed spheroid structures in vitro. After subcutaneous injection into nude mice, these spheroids formed tumors with growth characteristics similar to those of crypt-derived spheroids. Tumors from both crypt and villus-derived spheres were highly proliferative and strongly expressed Lgr5. Lysozyme⁺ Paneth cells were detectable in tumors of either origin, further underscoring the pluripotent potential. After separating Lgr5⁺ and Lgr5⁻ cells from villus-derived spheres, both populations retained the capacity to initiate subcutaneous tumor growth. Tumors from initially Lgr5⁻ populations re-expressed Lgr5. Taken together, these results strongly support the notion that villus cells can re-acquire cancer stem cell properties by dedifferentiation when Wnt signaling is elevated in a NFκB-dependent manner.

Crypt-like foci could theoretically have originated from the Lgr5⁺ cells at the base of the crypt and have moved upward. To prove that dedifferentiated villus cells can initiate tumorigenesis in vivo, Schwitalla et al. generated a mouse that allowed recombination in Lgr5⁻ cells only. When β-catenin activation was enhanced, tumor initiation commenced, recapitulating the phenotype that had been observed in the organoid culture system. These polyps were highly proliferative and characterized by elevated NFκB activation. Most importantly, tumors re-expressed stem cell marker Lgr5 and displayed increased expression of genes encoding members of the “stem cell signature”, providing direct genetic evidence for dedifferentiation of Lgr5⁻ IEC in vivo.⁷

Cell-type plasticity can be observed during epithelial–mesenchymal transition (EMT) and has been suggested as important prerequisite for the metastatic spread of solid tumors.⁸ Recent data suggest that dedifferentiation of non-stem cells triggered by signals from the inflammatory microenvironment may also lead to the generation of cancer stem cells.⁹ So far, in vivo evidence for the existence of dedifferentiation was limited. Here, Schwitalla et al. demonstrate in a genetic model of intestinal tumor initiation that epithelial non-stem cells can re-express stem cell markers and can be converted into tumor-initiating cells. This phenomenon is strictly dependent on the degree of Wnt activation and can only be observed when Wnt signaling is markedly elevated which is often observed in the context of chronic inflammation. Therefore, chronic inflammation can contribute considerably to dedifferentiation of IEC and cancer development with NFκB as keystone.

Two models for the histopathogenesis of colorectal cancer have been proposed: a “top-down” model suggesting that dysplastic cells spread laterally and downward to form new crypts and a “bottom-up” model that is based on transformation and expansion of crypt stem cells.^10,11 Recent data show that Lgr5⁺ cells comprise the tumor-initiating cell population.¹² Under physiological circumstances these cells reside exclusively at the bottom of intestinal crypts, therefore the “bottom-up” model seemed to represent the prevailing concept.¹³ Schwitalla et al. now provide genetic evidence that Lgr5⁻ enterocytes have the potential to dedifferentiate and to re-acquire stem cell properties including Lgr5 expression, supporting a “top-down” model. Therefore, both models seem to not exclude each other. Rather, tumor-initiating mutations can occur in both Lgr5⁺ crypt stem cells or in more differentiated Lgr5⁻ cell since these initially negative cells can dedifferentiate and re-express Lgr5.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

10.4161/cbt.27629