Tissue-Intrinsic Tumor Hotspots: Terroir for Tumorigenesis
Authors: Yoichiro Tamori, Wu-Min Deng
Journal: Trends in cancer (2017)

Tumor initiation depends on tissue-intrinsic local cytoarchitectures and endogenous local growth signaling activities in Drosophila imaginal-disc epithelia. Polarity-deficient protumor cells bearing mutant neoplastic tumor-suppressor genes are frequently eliminated from epithelial tissues through cell competition-dependent apoptosis or spindle misorientation-induced delamination. Local cytoarchitecture of epithelial tissues is involved in determining delamination direction of polarity-deficient protumor cells. Direction of delamination from epithelial layer has a decisive role for the life-or-death fate of protumor cells. Epithelial tissues are highly organized systems with a remarkable homeostatic ability to maintain morphology through regulation of cellular proliferation and tissue integrity. This robust self-organizing system is progressively disrupted during tumor development. Recent studies of conserved tumor-suppressor genes in Drosophila showed how protumor cells deviate from the robustly organized tissue microenvironment to take the first steps into becoming aggressive tumors. Here we review the 'tumor hotspot' hypothesis that explains how the tissue-intrinsic local microenvironment has a pivotal role in the initial stage of tumorigenesis in Drosophila epithelia and discuss comparable mechanisms in mammalian tissues. Epithelial tissues are highly organized systems with a remarkable homeostatic ability to maintain morphology through regulation of cellular proliferation and tissue integrity. This robust self-organizing system is progressively disrupted during tumor development. Recent studies of conserved tumor-suppressor genes in Drosophila showed how protumor cells deviate from the robustly organized tissue microenvironment to take the first steps into becoming aggressive tumors. Here we review the 'tumor hotspot' hypothesis that explains how the tissue-intrinsic local microenvironment has a pivotal role in the initial stage of tumorigenesis in Drosophila epithelia and discuss comparable mechanisms in mammalian tissues. Throughout life span, a vast number of cells continually experience various stressors and mutagens from exogenous and endogenous sources, many of which cause genetic mutations in the cell. If the mutation involves activation of an oncogene, or inactivation of a tumor-suppressor gene, the mutant cell could act as a cancer 'seed'. In fact, recent studies of healthy human tissues showed that somatic mutations including nucleotide substitutions and chromosomal anomalies increase with age, suggesting that cells carrying cancer-causing mutations accumulate over time in various types of tissue [1Jacobs K.B. et al.Detectable clonal mosaicism and its relationship to aging and cancer.Nat. 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These studies analyzed conserved neoplastic tumor-suppressor genes (nTSGs) (see Glossary) in the Drosophila wing imaginal-disc epithelia and showed that tumor initiation depends on tissue-intrinsic local cytoarchitectures and endogenous local growth signaling activities, and that tumors consistently originate in a specific tissue region. Here we present a review of the recently proposed 'tumor hotspot' hypothesis that explains how 'terroir' has a decisive role in the initial stages of tumorigenesis. Epithelial tissues are composed of epithelial cells communicating with their neighbors and environment to regulate cellular proliferation and tissue integrity. This highly organized system has a remarkable homeostatic ability to maintain tissue integrity and organ size through turnover of its units and repair of damaged parts after injury. During cancer development, however, this robust self-organizing system is progressively disrupted. 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It was observed that some of those apoptotic nTSG mutant cells stayed in the epithelial layer, suggesting that apoptosis was not caused by basal extrusion [16Tamori Y. et al.Involvement of Lgl and Mahjong/VprBP in cell competition.PLoS Biol. 2010; 8: e1000422Crossref PubMed Scopus (135) Google Scholar]. In the genetically mosaic epithelia, apoptosis was mostly detected in nTSG mutant cells that were at the clone boundary, suggesting that the presence of adjacent normal cells triggered competition-dependent apoptosis of nTSG mutant cells [15Igaki T. et al.Intrinsic tumor suppression and epithelial maintenance by endocytic activation of Eiger/TNF signaling in Drosophila.Dev. 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These studies suggest that elimination of protumor cells such as nTSG mutant cells from normal tissues is an evolutionarily conserved epithelial self-defense mechanism against cancer [31Kajita M. Fujita Y. EDAC: epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals.J. Biochem. 2015; 158: 15-23Crossref PubMed Scopus (70) Google Scholar]. The elimination of nTSG mutant cells in Drosophila imaginal discs might also be explained by a different mechanism. Epithelial tissues are composed of apicobasally polarized cells. To maintain the morphological architecture of the epithelial sheet wherein epithelial cells are arranged in a plane, the direction of cell division needs to be parallel to the plane of the epithelial sheet. Several genetic analysis and in vivo live-imaging studies of nTSGs in Drosophila showed that the protein products of lgl, scrib, and dlg have a key role in determining the planar orientation of the mitotic spindle that interacts with mitotic apparatuses in proliferating epithelial cells [32Nakajima Y.-I. et al.Epithelial junctions maintain tissue architecture by directing planar spindle orientation.Nature. 2013; 500: 359-362Crossref PubMed Scopus (142) Google Scholar, 33Bell G.P. et al.Aurora kinases phosphorylate Lgl to induce mitotic spindle orientation in Drosophila epithelia.Curr. Biol. 2015; 25: 61-68Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 34Carvalho C.A. et al.Aurora A triggers Lgl cortical release during symmetric division to control planar spindle orientation.Curr. Biol. 2015; 25: 53-60Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. 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Conditional deletion of Scrib in mice mammary gland results in excess growth of atypical luminal cells, and development of ductal and alveolar hyperplasia associated with aberrant spindle orientation [36Godde N.J. et al.Scribble modulates the MAPK/Fra1 pathway to disrupt luminal and ductal integrity and suppress tumour formation in the mammary gland.PLoS Genet. 2014; 10: e1004323Crossref PubMed Scopus (44) Google Scholar]. These findings indicate an evolutionarily conserved function for nTSG proteins in maintaining mitotic spindle orientation. When nTSG genes are disrupted, Drosophila imaginal-disc cells show fluctuation in the direction of mitotic spindles and abnormal planar orientation, which in turn cause misoriented cell division orthogonal to the plane of the epithelium [32Nakajima Y.-I. et al.Epithelial junctions maintain tissue architecture by directing planar spindle orientation.Nature. 2013; 500: 359-362Crossref PubMed Scopus (142) Google Scholar]. Consequently, basally located daughter cells delaminate from the epithelial layer and undergo apoptosis (Figure 2). This spindle misorientation-induced elimination of nTSG mutant cells is triggered by live-cell delamination, since apoptosis of nTSG mutant cells was observed in the basal side of epithelial layer after delamination. Although delamination-induced cell death differs from cell competition, which actively eliminates loser cells, most of protumor cells are also eliminated from the tissue. Both cell competition and spindle misorientation-induced delamination are involved in eliminating deleterious protumor cells, and thus decrease the possibility of tumor formation in epithelial tissues. Interestingly, we have shown in Drosophila wing imaginal discs that loss-of-nTSG-induced tumor formation is dependent on the location of the protumor cells. Tumors induced by nTSG knockdown were always found initially in the peripheral 'hinge' region and never observed in the central 'pouch' region of the disc epithelium [9Tamori Y. et al.Epithelial tumors originate in tumor hotspots, a tissue-intrinsic microenvironment.PLoS Biol. 2016; 14: e1002537Crossref PubMed Scopus (53) Google Scholar] (Figure 3A,B). The tumorigenic potential of nTSG-knockdown cells therefore depends on their local environment in the epithelial tissue. The central pouch region acts like a 'tumor coldspot' where protumor cells do not show dysplastic overgrowth, while the peripheral hinge region behaves as a 'tumor hotspot' [9Tamori Y. et al.Epithelial tumors originate in tumor hotspots, a tissue-intrinsic microenvironment.PLoS Biol. 2016; 14: e1002537Crossref PubMed Scopus (53) Google Scholar]. A key difference between nTSG-knockdown cells located in coldspots and hotspots is the direction of delamination. In the hotspot hinge area, protumor cells delaminate from the apical side and show dysplastic growth. By contrast, in coldspot areas, nTSG-knockdown cells delaminate from the basal side of epithelial layer and undergo apoptosis (Figure 3C). Cell competition-induced apoptosis, however, occurs in both the pouch coldspot and hinge hotspot regions. These findings suggesting that some nTSG-knockdown cells in the hinge area survive cell competition and delaminate from the apical side prompted a close examination of the tissue and cellular organization between these different regions of the wing disc. One key difference is the morphology of cells composing the pseudostratified monolayer, such that cells in the flat wing pouch coldspot are elongated along their apical–basal axis, whereas cells in the folded hinge hotspot regions are shorter [9Tamori Y. et al.Epithelial tumors originate in tumor hotspots, a tissue-intrinsic microenvironment.PLoS Biol. 2016; 14: e1002537Crossref PubMed Scopus (53) Google Scholar]. A follow-up analysis using transmission electron micrographs revealed marked differences in the basal side of the epithelial layer: (i) in the valley-folded hotspot, cellular membranes display a complicated set of bends at the basal side, whereas in the coldspot they appear straight along the apical–basal axis; (ii) hotspot cells show filopodia-like protrusions at the basal surface that elongate laterally and intertwine intricately with the protrusions from neighboring cells; and (iii) the basement membrane composed of approximately ten thin laminae is organized loosely in the coldspot, but aligned tightly in the hotspot. These basal-specific structures of the hotspot are hypothesized to prevent delamination of protumor cells from its basal surface [9Tamori Y. et al.Epithelial tumors originate in tumor hotspots, a tissue-intrinsic microenvironment.PLoS Biol. 2016; 14: e1002537Crossref PubMed Scopus (53) Google Scholar] (Figure 4). Why do apically delaminated protumor cells survive and proliferate, but basally delaminated ones die? One plausible reason for the death of the basally delaminated cells is apoptosis induced by tumor necrosis factor (TNF). TNF, a ligand of the death receptors, functions as an extracellular signal to activate proapoptotic cell surface receptors [37Sedger L.M. McDermott M.F. TNF and TNF-receptors: from mediators of cell death and inflammation to therapeutic giants – past, present and future.Cytokine Growth Factor Rev. 2014; 25: 453-472Crossref PubMed Scopus (532) Google Scholar]. In the Drosophila imaginal discs, apoptosis of nTSG mutant protumor cells is induced by Eiger, the Drosophila homolog of TNF-α, which is produced by circulating hemocytes recruited to tumor tissues [38Cordero J.B. et al.Oncogenic Ras diverts a host TNF tumor suppressor activity into tumor promoter.Dev. Cell. 2010; 18: 999-1011Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar]. 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Luminal translocation thus allows oncogene-expressing cells to evade the suppressive epithelial environment and unleash their tumorigenicity, yet translocation alone is not sufficient to induce luminal outgrowth. But why can apically delaminated nTSG-knockdown cells survive and show tumorigenic overgrowth in the luminal region in the absence of an oncogenic mutation? It turns out that the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is endogenously active specifically in the tumor-hotspot region of developing wing imaginal discs [9Tamori Y. et al.Epithelial tumors originate in tumor hotspots, a tissue-intrinsic microenvironment.PLoS Biol. 2016; 14: e1002537Crossref PubMed Scopus (53) Google Scholar]. Dysregulation of JAK/STAT signaling has been implicated in diverse types of human cancers [47Thomas S.J. et al.The role of JAK/STAT signalling in the pathogenesis, prognosis and treatment of solid tumours.Br. J. 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Endogenous activity of the JAK/STAT pathway is high in the medial fold, weak in the proximal fold, and barely detectable in the distal fold. Indeed, dysplastic tumor growth induced by nTSG knockdown was mostly observed in the medial fold (Figure 3C). Further analysis showed that depletion of STAT92E, the Drosophila homolog of mammalian STAT3 and STAT5 [53Hou X.S. et al.Marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the mammalian STATs.Cell. 1996; 84: 411-419Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 54Yan R. et al.Identification of a Stat gene that functions in Drosophila development.Cell. 1996; 84: 421-430Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar], blocked the dysplastic tumor growth in nTSG-knockdown cells, indicating that STAT activation is necessary for nTSG-knockdown-induced tumorigenesis. Moreover, overactivation of STAT in nTSG-knockdown cells in tumor hotspots, including the distal fold of the dorsal hinge, dramatically enhanced tumor size when compared with nTSG-knockdown-induced tumors without STAT overactivation from the same time point postclone induction. These results indicate that nTSG-knockdown cells exploit local endogenous activity of the JAK/STAT pathway to survive and proliferate [9Tamori Y. et al.Epithelial tumors originate in tumor hotspots, a tissue-intrinsic microenvironment.PLoS Biol. 2016; 14: e1002537Crossref PubMed Scopus (53) Google Scholar] (Figure 4). Similar to reports in mammalian epithelial cells [55Rossi O. et al.Vectorial secretion of interleukin-8 mediates autocrine signalling in intestinal epithelial cells via apically located CXCR1.BMC Res. Notes. 2013; 6: 431Crossref PubMed Scopus (26) Google Scholar, 56Healy L.L. et al.Polarized epithelial cells secrete interleukin 6 apically in the bovine endometrium.Biol. 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In vertebrate epithelia, instead of basal extrusion, oncogenic protumor cells predominantly undergo apical extrusion into the lumen, which basically eliminates protumor cells [27Hogan C. et al.Characterization of the interface between normal and transformed epithelial cells.Nat. Cell Biol. 2009; 11: 460-467Crossref PubMed Scopus (250) Google Scholar, 58Slattum G. et al.P115 RhoGEF and microtubules decide the direction apoptotic cells extrude from an epithelium.J. Cell Biol. 2009; 186: 693-702Crossref PubMed Scopus (104) Google Scholar, 59Slattum G.M. Rosenblatt J. nrc3767.Nat. Rev. Cancer. 2014; 14: 495-501Crossref PubMed Scopus (81) Google Scholar]. Defects in apical extrusion could enhance basal extrusion and survival of tumor cells, which enable their invasion beneath the epithelium [60Gu Y. et al.Defective apical extrusion signaling contributes to aggressive tumor hallmarks.Elife. 2014; 4: 491Google Scholar, 61Gudipaty S.A. Rosenblatt J. 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Outgrowth of single oncogene-expressing cells from suppressive epithelial environments.Nature. 2012; 482: 410-413Crossref PubMed Scopus (180) Google Scholar]. Taken together, the direction of protumor cell extrusion and its implication in tumor cell survival are highly context dependent. What matters is whether the protumor cells will enter a suitable microenvironment for their survival and growth. In pathological histology, it is known that tumors frequently arise in transition zones where two different types of epithelial tissue meet, resulting in the appearance of a distinct abrupt transition, which can be found in numerous locations within various tissues such as the junction of the cervix with the uterus and the junction of the esophagus and the stomach [63Mcnairn A.J. Guasch G. Epithelial transition zones: merging microenvironments, niches, and cellular transformation.Eur. J. Dermatol. 2011; 21: 21-28Crossref PubMed Scopus (58) Google Scholar]. In addition, it is also known that epithelial carcinogenesis frequently arises from metaplasia, where one type of normal cell layer is displaced by cells of another type that are normally resident in a different organ [64Delvenne P. et al.Epithelial metaplasia: an inadequate environment for antitumour immunity?.Trends Immunol. 2004; 25: 169-173Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar]. In fact, metaplasia is most frequent in epithelial transition zones. Even though these metaplastic cells have a normal appearance, metaplasia is considered an early step in the development of carcinomas. 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Intriguingly, even in wild-type mice, the epithelium of the transition zone in the anus intrinsically shows many features reminiscent of hyperproliferative epidermis including aberrant expression of differentiation markers, enhanced Ras-mitogen-activated protein kinase signaling, and locally increased inflammation [66Guasch G. et al.Loss of TGFbeta signaling destabilizes homeostasis and promotes squamous cell carcinomas in stratified epithelia.Cancer Cell. 2007; 12: 313-327Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 67Wakefield L.M. Stuelten C. Keeping order in the neighborhood: new roles for TGFbeta in maintaining epithelial homeostasis.Cancer Cell. 2007; 12: 293-295Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar]. Future studies will determine if the transition zones in mammalian tissues have similar functions to the tumor hotspots found in Drosophila imaginal discs. Although a number of causative genetic backgrounds of tumor development have been discovered, still little is known about how protumor cells deviate from the robustly organized tissue environment to take the first steps in their evolution into aggressive tumors. The study of tumor initiation in Drosophila imaginal discs suggests that two different processes are closely involved in tumorigenesis by nTSG defects in tumor hotspots: delamination of surviving protumor cells and proliferation outside of the epithelial layer promoted by endogenous JAK/STAT activity [9Tamori Y. et al.Epithelial tumors originate in tumor hotspots, a tissue-intrinsic microenvironment.PLoS Biol. 2016; 14: e1002537Crossref PubMed Scopus (53) Google Scholar]. Apical delamination of protumor cells is caused by intrinsic local cytoarchitectures specifically observed in the basal side of hotspots. Tumor-promoting local JAK/STAT activity is also endogenously patterned in the tissue. These facts suggest that tissue-intrinsic factors are responsible for the fate of protumor cells. Although it is still unclear how these two factors are related to each other, it is possible that coexistence of the two causal factors creates a local 'terroir' suitable for the survival and growth of protumor seeds. It is also possible that protumor cells may utilize additionally occurring mutations to create hotspot-like terroir by themselves during multistep tumor progression. To determine the significance of local terroir for tumorigenesis in humans, first we need to ask whether tumor hotspots exist in human epithelial tissues (see Outstanding Questions). The transition zones in mammalian tissues are reminiscent of tumor hotspots found in Drosophila. Furthermore, given the fact that tumor hotspots are formed in small local areas (smaller than 50 cells in diameter) in Drosophila imaginal-disc epithelia, it is possible that a substantial number of local 'terroir' exist in human epithelial tissues. Because human tumors are composed of a billion or more cells when clinically detected [68Moreno E. Is cell competition relevant to cancer?.Nat. Rev. Cancer. 2008; 8: 141-147Crossref PubMed Scopus (149) Google Scholar], the specific location where the tumor arose is difficult to determine. However, it might be possible to quantify the regional frequency of tumorous lesion in an organ. Examination of specific cytoarchitectures and endogenous activities of oncogenic or inflammatory signaling pathways in regions where primary tumors are frequently observed will help us identify tumor hotspots in human tissues, which may allow better prediction and earlier detection of malignancies, and ultimately lead to preventive therapies. Future studies to identify the causal factors that facilitate formation of tumor hotspots in various types of tissues or to clarify the behaviors of different types of protumor cells in tumor hotspots will lead to a better understanding of tumor initiation.Outstanding QuestionsHow are the two causal factors (local cytoarchitecture and endogenous JAK/STAT activity) involved in formation of tissue-intrinsic tumor hotspots in Drosophila imaginal-discs epithelia related to each other?Does a tissue-intrinsic microenvironment similar to the tumor hotspot found in Drosophila imaginal-disc epithelia also exist in human tissues?Is the regional frequency of tumorous lesions varied in a human organ? How are the two causal factors (local cytoarchitecture and endogenous JAK/STAT activity) involved in formation of tissue-intrinsic tumor hotspots in Drosophila imaginal-discs epithelia related to each other? Does a tissue-intrinsic microenvironment similar to the tumor hotspot found in Drosophila imaginal-disc epithelia also exist in human tissues? Is the regional frequency of tumorous lesions varied in a human organ? We thank G. Calvin for critical reading of the manuscript. This work was supported by grants from National Science FoundationIOS-1052333 and National Institutes of HealthR01GM072562 to W.-M.D. and JSPS KAKENHI Grant Numbers 26891025 and 15H01500 and The Uehara Memorial Foundation Research Grant to Y.T. fitter cells kill and eliminate less-fit neighbors in a tissue. structures and arrangements of cells in a tissue. peripheral region of wing imaginal discs, which develop into the hinge of wing. developing epithelial tissues in the larva of insects. tumor-suppressor genes, lethal giant larvae (lgl), discs large (dlg), and scribble (scrib), that control cell polarity and proliferation in epithelia and neuroblasts. improper alignment of mitotic spindles, which induces a misdirected cell division. environmental conditions including soil and climate in which a plant is grown. regions where protumor cells do not undergo tumorigenesis. tissue-intrinsic microenvironment where tumorigenesis preferentially occur. center region of wing imaginal discs, which develop into the wing blade.