Julia B. Cordero

ROS Production and NF-κB Activation Triggered by RAC1 Facilitate WNT-Driven Intestinal Stem Cell Proliferation and Colorectal Cancer Initiation

Summary The Adenomatous Polyposis Coli (APC) gene is mutated in the majority of colorectal cancers (CRCs). Loss of APC leads to constitutively active WNT signaling, hyperproliferation, and tumorigenesis. Identification of pathways that facilitate tumorigenesis after APC loss is important for therapeutic development. Here, we show that RAC1 is a critical mediator of tumorigenesis after APC loss. We find that RAC1 is required for expansion of the LGR5 intestinal stem cell (ISC) signature, progenitor hyperproliferation, and transformation. Mechanistically, RAC1-driven ROS and NF-κB signaling mediate these processes. Together, these data highlight that ROS production and NF-κB activation triggered by RAC1 are critical events in CRC initiation.

Oncogenic Ras Diverts a Host TNF Tumor Suppressor Activity into Tumor Promoter

The roles of inflammatory cytokines and the immune response in cancer remain paradoxical. In the case of tumor necrosis factor (TNF), there is undisputed evidence indicating both protumor and antitumor activities. Recent work in Drosophila indicated that a TNF-dependent mechanism eliminates cells deficient for the polarity tumor suppressors dlg or scrib. In this study, however, we show that in tumors deficient for scrib that also expressed the Ras oncoprotein, the TNF signal was diverted into a protumor signal that enhanced tumor growth through larval arrest and stimulated invasive migration. In this case, TNF promoted malignancy and was detrimental to host survival. TNF was expressed at high levels by tumor-associated hemocytes recruited from the circulation. The expression of TNF by hemocytes was both necessary and sufficient to trigger TNF signaling in tumor cells. Our evidence suggests that tumors can evolve into malignancy through oncogenic Ras activation and the hijacking of TNF signaling.

MicroRNA-135b Promotes Cancer Progression by Acting as a Downstream Effector of Oncogenic Pathways in Colon Cancer

Summary MicroRNA deregulation is frequent in human colorectal cancers (CRCs), but little is known as to whether it represents a bystander event or actually drives tumor progression in vivo. We show that miR-135b overexpression is triggered in mice and humans by APC loss, PTEN/PI3K pathway deregulation, and SRC overexpression and promotes tumor transformation and progression. We show that miR-135b upregulation is common in sporadic and inflammatory bowel disease-associated human CRCs and correlates with tumor stage and poor clinical outcome. Inhibition of miR-135b in CRC mouse models reduces tumor growth by controlling genes involved in proliferation, invasion, and apoptosis. We identify miR-135b as a key downsteam effector of oncogenic pathways and a potential target for CRC treatment.

mTORC1 mediated translational elongation limits intestinal tumour initiation and growth

Inactivation of APC is a strongly predisposing event in the development of colorectal cancer, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1–S6K–eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer.

Inducible progenitor-derived Wingless regulates adult midgut regeneration in Drosophila

The ability to regenerate following stress is a hallmark of self‐renewing tissues. However, little is known about how regeneration differs from homeostatic tissue maintenance. Here, we study the role and regulation of Wingless (Wg)/Wnt signalling during intestinal regeneration using the Drosophila adult midgut. We show that Wg is produced by the intestinal epithelial compartment upon damage or stress and it is exclusively required for intestinal stem cell (ISC) proliferation during tissue regeneration. Reducing Wg or downstream signalling components from the intestinal epithelium blocked tissue regeneration. Importantly, we demonstrate that Wg from the undifferentiated progenitor cell, the enteroblast, is required for Myc‐dependent ISC proliferation during regeneration. Similar to young regenerating tissues, ageing intestines required Wg and Myc for ISC hyperproliferation. Unexpectedly, our results demonstrate that epithelial but not mesenchymal Wg is essential for ISC proliferation in response to damage, while neither source of the ligand is solely responsible for ISC maintenance and tissue self‐renewal in unchallenged tissues. Therefore, fine‐tuning Wnt results in optimal balance between the ability to respond to stress without negatively affecting organismal viability.

Integrin signalling regulates YAP and TAZ to control skin homeostasis.

ABSTRACT The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib. Summary: In the squamous epithelium of the mouse skin, the transcription factors YAP and TAZ are regulated by integrin-Src signalling, and are required for proliferation of skin stem/progenitor cells.

Non-autonomous crosstalk between the Jak/Stat and EGFR pathways mediates Apc1-driven intestinal stem cell hyperplasia in the Drosophila adult midgut

Inactivating mutations within adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, are responsible for most sporadic and hereditary forms of colorectal cancer (CRC). Here, we use the adult Drosophila midgut as a model system to investigate the molecular events that mediate intestinal hyperplasia following loss of Apc in the intestine. Our results indicate that the conserved Wnt target Myc and its binding partner Max are required for the initiation and maintenance of intestinal stem cell (ISC) hyperproliferation following Apc1 loss. Importantly, we find that loss of Apc1 leads to the production of the interleukin-like ligands Upd2/3 and the EGF-like Spitz in a Myc-dependent manner. Loss of Apc1 or high Wg in ISCs results in non-cell-autonomous upregulation of upd3 in enterocytes and subsequent activation of Jak/Stat signaling in ISCs. Crucially, knocking down Jak/Stat or Spitz/Egfr signaling suppresses Apc1-dependent ISC hyperproliferation. In summary, our results uncover a novel non-cell-autonomous interplay between Wnt/Myc, Egfr and Jak/Stat signaling in the regulation of intestinal hyperproliferation. Furthermore, we present evidence suggesting potential conservation in mouse models and human CRC. Therefore, the Drosophila adult midgut proves to be a powerful genetic system to identify novel mediators of APC phenotypes in the intestine.

A role for wingless in an early pupal cell death event that contributes to patterning the Drosophila eye

Programmed cell death (PCD) is utilized in a wide variety of tissues to refine structure in developing tissues and organs. However, little is understood about the mechanisms that, within a developing epithelium, combine signals to selectively remove some cells while sparing essential neighbors. One popular system for studying this question is the developing Drosophila pupal retina, where excess interommatidial support cells are removed to refine the patterned ommatidial array. In this paper, we present data indicating that PCD occurs earlier within the pupal retina than previously demonstrated. As with later PCD, this death is dependent on Notch activity. Surprisingly, altering Drosophila Epidermal Growth Factor Receptor or Ras pathway activity had no effect on this death. Instead, our evidence indicates a role for Wingless signaling to provoke this cell death. Together, these signals regulate an intermediate step in the selective removal of unneeded interommatidial cells that is necessary for a precise retinal pattern.

Local control of intestinal stem cell homeostasis by enteroendocrine cells in the adult Drosophila midgut.

Summary Background Enteroendocrine cells populate gastrointestinal tissues and are known to translate local cues into systemic responses through the release of hormones into the bloodstream. Results Here we report a novel function of enteroendocrine cells acting as local regulators of intestinal stem cell (ISC) proliferation through modulation of the mesenchymal stem cell niche in the Drosophila midgut. This paracrine signaling acts to constrain ISC proliferation within the epithelial compartment. Mechanistically, midgut enteroendocrine cells secrete the neuroendocrine hormone Bursicon, which acts—beyond its known roles in development—as a paracrine factor on the visceral muscle (VM). Bursicon binding to its receptor, DLGR2, the ortholog of mammalian leucine-rich repeat-containing G protein-coupled receptors (LGR4-6), represses the production of the VM-derived EGF-like growth factor Vein through activation of cAMP. Conclusions We therefore identify a novel paradigm in the regulation of ISC quiescence involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells.

Dynamic decapentaplegic signaling regulates patterning and adhesion in the Drosophila pupal retina.

The correct organization of cells within an epithelium is essential for proper tissue and organ morphogenesis. The role of Decapentaplegic/Bone morphogenetic protein (Dpp/BMP) signaling in cellular morphogenesis during epithelial development is poorly understood. In this paper, we used the developing Drosophila pupal retina - looking specifically at the reorganization of glial-like support cells that lie between the retinal ommatidia - to better understand the role of Dpp signaling during epithelial patterning. Our results indicate that Dpp pathway activity is tightly regulated across time in the pupal retina and that epithelial cells in this tissue require Dpp signaling to achieve their correct shape and position within the ommatidial hexagon. These results point to the Dpp pathway as a third component and functional link between two adhesion systems, Hibris-Roughest and DE-cadherin. A balanced interplay between these three systems is essential for epithelial patterning during morphogenesis of the pupal retina. Importantly, we identify a similar functional connection between Dpp activity and DE-cadherin and Rho1 during cell fate determination in the wing, suggesting a broader link between Dpp function and junctional integrity during epithelial development.