REVIEW ISSN 1598-9100(Print) ISSN 2288-1956(Online) http://dx.doi.org/10.5217/ir.2013.11.2.85 Intest Res 2013;11(2):85-91 Stem Cells in Colorectal Cancer: New Potential Therapeutic Target Tae Il Kim Department of Internal Medicine and Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Korea Within the crypts of the intestinal mucosa, intestinal epithelium is a permanently renewing tissue, the architecture of which is maintained by the ability of the intestinal stem cells to self-renew and to generate a hierarchy of proliferative and differentiated cells. In the hierarchical structure of intestinal epithelia, the balance between proliferation and cell death is important for homeostasis. This unique structure of intestinal mucosa, crypt axis, is supported by micro-environmental factors, and the disruption of the homeostasis of the crypt axis can develop colorectal neoplasia. Recent evidence suggests that colorectal cancer may arise from mutated colorectal stem or progenitor cells termed colorectal cancer stem cells (CSC) or initiating cells because of their exclusive ability to sustain tumor formation. Colorectal CSC have been identified based on the expression of cell surface markers such as CD133, CD44 and CD166, and these cells have stem/progenitor cell properties, the ability to self-renew, differentiate, and proliferate indefinitely to drive continuous expansion of the malignant cell population. The CSCs, in limited number within the bulk of the tumor, may account for their capability of escaping conventional therapies, thus leading to disease relapse and metastasis. To overcome these malignant features of cancer, the researchers emphasize the importance of better characterizing CSC to target the CSC. (Intest Res 2013;11:85-91) Key Words: Stem cells; Colorectal neoplasms; Therapy 서론 난치성질환의치료가능성을제시하면서줄기세포에대한연구가 많은주목을받고있으며, 재생의학 (regenerative medicine) 분야뿐 만아니라암질환의병태생리의이해에서부터치료에이르기까지여 러분야에기여하고있다. 모든장기에줄기세포와그로부터분화되 어최종기능을수행하는분화세포에이르는구성단위가있듯이, 장점 막도장선와 (intestinal crypt) 의구성단위로이루어져있다. 장선와내 줄기세포와분화세포, 그리고주위미세환경의상호작용에의한장 상피세포의항상성은매우잘조절되지만, 1 주정도의주기로빠르게 세포의이동, 분화, 사멸, 재생의과정이반복되는장선와에서이러한 조절의이상은암발생의위험과높은연관성을가진다. 정상장선와 와같이대장암에서도구성세포간의계층적분화과정이있고그중 Received March 11, 2013. Revised March 14, 2013. Accepted March 17, 2013. Correspondence to Tae Il Kim, Institute of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea. Tel: +82-2-2228-1965, Fax: +82-2- 393-6884, E-mail: taeilkim@yuhs.ac 심에는암줄기세포가있으며, 최근암극복과완치를위한치료표적으로서암줄기세포를표적으로하는많은연구가이루어지고있다. 정상장줄기세포의항상성유지및표지자정상장선와의항상성조절기전에대한이해는줄기세포표지자발굴과함께대장암의발생과진행에대한연구에많은기여를하고있다. 장점막은상피세포의증식, 분화와사멸이선와-융모축 (cryptvillus axis) 을따라순차적으로일어나는독특한구조를가지고있으며, 세포의순환이빠르고일정하며증식부위와분화부위가분리되어있으므로상피세포의줄기세포연구에좋은모델이되고있다. 소장상피의융모부위는충분히분화되어더이상증식할수없는세포로구성되어있으며, 영양소흡수및물리적방어벽으로서역할을한다. 선와에해당하는증식부위는빠르게증식하는미분화세포로구성되어세포순환을조절하고줄기세포의 niche 와상호관계를유지하고있으며, 주요증식세포는선와의하부에위치하게된다. 소장에서줄기세포는흡수장세포 (absorptive enterocyte), 점액분 Copyright 2013. Korean Association for the Study of Intestinal Diseases. All rights reserved. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Tae Il Kim Colorectal Cancer Stem Cell Fig. 1. Homeostasis of colon crypt and stem cell-regulating factors. Colon crypts are maintained by the ability of the intestinal stem cells to self-renew and to generate a hierarchy of proliferative and differentiated cells via complex signaling pathway, such as WNT, Notch and bone morphogenetic protein (BMP), and supported by microenvironments, including pericryptal myofibroblast and inflammatory cells. 비술잔세포 (goblet cell), 장내분비 (enteroendocrine) 세포, 그리고 Paneth 세포의 4가지상피세포로분화한다. 장세포, 술잔세포, 장내분비세포는융모의위쪽방향으로이동할수록분화하며, 융모끝에이르게되면떨어져나가면서세포사멸에이르게된다. Paneth 세포는줄기세포위치로부터선와의기저부쪽으로, 즉다른세포들의분화방향과는반대인아래쪽으로이동함에따라분화하게되며여기에는 EphB3 발현이관여한다고알려져있다. 1,2 대장점막에서는융모와 Paneth 세포가없다는점이다르고그외장선와의구조는비슷하다 (Fig. 1). 장줄기세포표지자가알려져있지않았던시기에장줄기세포의상대적위치는 bromo-deoxyuridine 및 3 H-thymidine labeling 을이용한실험에서표지된한쪽 DNA 가닥을계속유지하는줄기세포의특성을통해알려졌고, 이세포는장선와에서선와기저부로부터 4번째세포층에위치하여 +4 labeling retaining cell (LRC) 로알려져있다. 그리고선와형태형성 (crypt morphogenesis) 동안각선와에서하나의줄기세포가선택되어해당장상피선와를채우게되며, 이같은사실은다클론배아선와 (polyclonal embryonic crypt) 가단클론성인선와 (monoclonal adult crypt) 로바뀌는것으로확인되었다. 3 줄기세포연구에서가장중요한조건이특이적인줄기세포표지자인데, 최근장선와의기저부에존재하는장줄기세포표지자에대해 lineage-tracing 마우스실험을이용한몇가지주요한발견이있었다. Leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) 는장선와기저부의 Paneth 세포의사이에위치한 crypt base columnar 세포 (CBC cell) 를표지하는 WNT 신호전달의표적분자이고, 4 B lymphoma Mo-MLV insertion region 1 (BMI1) 과 telomerase reverse transcriptase (TERT) 는장선와의 +4 LRC 세포에서발현되는줄기세포표지자이다. 5-7 이들장줄기세포표지자들로대표되는장줄기세포들은위치및특성이서로다르므로장줄기세포도어느한가지표지자로설명할수없고여러다른특성의줄기세포가혼재되어있다고볼수있다. 여러특성상 +4 LRC 세포들이비활성상태의줄 기세포, CBC 세포가활성상태의줄기세포로생각되지만같은 +4 LRC 세포에서도 BMI1 발현세포와 TERT 발현세포는또다른특성을보이고 LGR5 세포와의상하관계도다르게나타나므로이들다른특성의장줄기세포간의상호작용, 상하관계, 그리고단클론성장선와를형성하게되는기전등에대해서는아직논란이많다. 8,9 그외에도 musashi 1 (Msi-1), OLFM4, ASCL2 등이오래전부터장줄기세포의다른표지자들로알려져있고, 10 최근 CD133 (Prominin-1), aldehyde dehydrogenase-1 (ALDH-1), doublecortin and CAM kinaselike 1 (DCAMKL-1), Ephrin B2 (EphB2), leucine-rich repeats and immunoglobulin-like domain1 (Lrig1) 등도주요장줄기세포표지자로보고되고있다. 11-14 대장암암화과정의암줄기세포및표지자와치료적용대장암의암화과정을대표하는 adenoma-carcinoma sequence 는대장암의발생과진행에있어여러주요유전자들의변이가축적되면서암이발생하고진행된다는잘정립된내용이지만, 그외미세환경의영향, 암종양내에세포간특성의다양성등과함께암줄기세포의개념이같이추가되었다. 즉종양내암세포의다양성에대한많은보고와함께, 이들중소수의특정세포군만이원래종양과동일특성의종양을형성할수있는능력이있다는내용으로암줄기세포의개념이시작되었다. 15,16 최근 BMI1, CD133, 그리고 LGR5 의 Cre recomninase 마우스를이용한연구에서장줄기세포에서유도된 adenomatous polyposis coli (APC) 유전자결손은마우스장샘종의발생을빠르게유도하였고, 줄기세포단계를지나분화된세포에발생한 APC 결손은일시적인변형소견이나느린샘종발생의소견을보였다. 7,11,17 이러한소견은분화단계의세포가아닌줄기세포에서의주요암관련유전자변이가암발생의궁극적세포표적이며, 암세포의기원이되는세포로서진정한암줄기세포가됨을의미한다. 그러나최근보고에의하면미세환 86 www.irjournal.org
다. 3 WNT 신호전달계억제를이용한대표적연구로서 WNT 수용체에 http://dx.doi.org/10.5217/ir.2013.11.2.85 Intest Res 2013;11(2):85-91 경에서유래한 hepatocyte growth factor (HGF), 또는염증성전사인자인 NFĸB 등의주요인자가이미줄기세포가아닌암세포를다시암줄기세포로역분화키는작용을할수있다. 18,19 이런의미에서대장암의여러다른특성들은암발생의기원세포의차이, 그리고해당기원세포에축적되는유전적변이의차이가주요원인일수있고, 이와더불어주위미세환경에의한영향도포함되어야할주요인자이다. 그러나아직암줄기세포의존재및중요성에대해여러이견이있으며, 이것은아직뚜렷한암줄기세포의공통표지자가부족하다는점에도원인이있다. 현재 CD133, CD166, CD44, CD24, ALDH-1, LGR5, epithelial cell adhesion molecule (EpCAM), BMI1, DCAM- KL-1 등의암줄기세포표지자가보고되었고, 12,13,15,16,20-22 이들표지자를이용한암줄기세포의특성이검증되고있다. 이런암줄기세포의개념을중요하게여기는이유는여러치료에저항성을보이는대표적인세포군이라는점과 23-25 재발암및암전이의주요원인이될수있다는점이있다 (Fig. 2). 18,23 여러연구에서일반적인항암치료후전체종양크기는감소하지만종양내암줄기세포의분획은오히려증가하는소견을보여치료저항성과재발암의주요원인이될수있음을보고하고있고, 암전이에중요한역할을하는 epithelial-mesenchymal transition (EMT) 현상이암줄기세포에서뚜 A B 렷하게증가됨이알려져있다. 26,27 따라서암줄기세포를표적으로하는치료는암의궁극적극복을가능하게하는방법중의하나가될수있다. 그러나많은암줄기세포표지자가정상줄기세포표지자와중복되므로단순한표지자를이용한표적치료에는한계가있을것으로생각된다. 뒤에서언급될장선와내에서정상줄기세포의증식과분화에관련된여러인자및신호전달계에대한이해를통해암줄기세포의자가재생증식을억제할방법이제안되고있으며, 정상줄기세포의유지, 증식및분화에서와유사한신호전달계가암줄기세포에서도작용하므로 WNT, Notch, bone morphogenetic protein (BMP) 등의신호전달물질을조절하고, 28,29 미세환경의영향을조절함으로써 18 암줄기세포의증식을억제하려는연구들이진행되고있다. 이와더불어암줄기세포의분화촉진을통하여줄기세포의특성을제거함으로써일반항암치료에대한반응을높이는방법이나, 이러한약제들을기존의항암제와같이병용투여하여치료저항성을극복하려는방법등이시도되고있다 (Fig. 2). 30-32 장줄기세포유지를위한주요신호전달줄기세포의유지와증식및분화를위해서는매우복잡한신호전달체계가필요하여, 줄기세포자체에의한조절뿐만아니라 niche 등의주위환경으로부터의신호전달역시매우중요한역할을할것으로알려져있다. 정상장줄기세포조절에관여하는많은인자및신호전달계가암줄기세포조절에서도주요인자로서관여하고있으므로정상장줄기세포조절인자에대한최근의연구발전은암줄기세포조절에대한연구에도많은공헌을하고있다. 현재까지알려져있는장줄기세포및장선와형성관련신호전달체계로서 WNT, Hedgehog (Hh), Notch, BMP 등이알려져있다 (Fig. 1). 1. WNT 신호와세포증식 C WNT 신호는 WNT 신호전달계의하나인 APC 유전자의변이가대장암발생에중요하게관여하므로위장관에서많이연구되었으며, 배아발달, 세포의항상성유지그리고여러질환의발생에관여하고있다. 특히장에서줄기세포유지및증식과분화에서중요한역할을한 Fig. 2. Concept of cancer stem cell (CSC)-targeted therapy. Conventional chemotherapy does not target cancer stem cells (red), which is resistant to chemo and radiation therapy. The tumor is reduced in size in the short term, but eventually relapses driven by cancer stem cells (A). When CSC-targeted therapy (B) or CSC-differentiating therapy (C) is combined with conventional therapy, tumor will progressively exhaust its growth potential. 결합하여 WNT 신호를억제하는 Dickkopf 를과발현시키거나 TCF4 를결손시킨마우스에서선와내증식감소와함께선와결손, 융모감소및융모위축의소견을보여, WNT 신호가장관내선와의증식구간에서지속적인세포증식에중요역할을함을보여준다. 33,34 WNT 신호의과발현을이용한연구로서 APC 결손의조건적유발에의한마우스실험에서 APC 결손유발직후선와의세포증식구간이확장되고분화및세포이동이억제되었으나, 융모에서는특별한형태학적변화가없었다. 35,36 그리고정상적으로선와기저부에서 Paneth 세포의위치와연관되어발현되는 EphB3 가 APC 결손후위쪽으로확장되어증식구간이확장된소견을보였다. 35,36 www.irjournal.org 87
Tae Il Kim Colorectal Cancer Stem Cell 또한 WNT 활성인자인 R-spondin1 은장줄기세포군의구간을확장시키고, 37 최근연구결과에의하면줄기세포표지자인 LGR5 를발현하는 CBC 세포옆에위치하는 Paneth 세포가줄기세포의유지와증식에관여하는 WNT3a 의주요한공급세포로밝혀졌다. 38 이와같이 WNT 신호에관련된많은인자들은암줄기세포유지및증식에관여하고있으므로암줄기세포억제를위한주요치료표적의하나이다. 39 또한 WNT 신호의근원지로서주위 niche 및미세환경이많이관여하므로미세환경조절또한중요한표적이다. 2. Notch 신호와세포증식및분화줄기세포로부터분화에이르는과정은줄기세포의분화프로그램과주위환경으로부터의여러신호에의해결정된다. Notch 신호의활성화는 hairy/enhancer of split (Hes) 유전자의발현을증가시키며, Hes-1 발현활성화는분비세포분화와관련된 Math-1 과 Neurogenin-3 와같은 basic helix-loop-helix (bhlh) 전사인자들의활성을억제한다. Notch 신호전달의중간매개체인 γ-secretase 를억제하면 Hes-1 억제에의해장점막내분비세포과다증식을유발하며, 40 발생배아에서 Hes-1 이결손된마우스는위와소장의내분비세포의조기발달을보이고장내술잔세포와장세포수가증가한다. 3,41 Notch 분자는 delta, jagged 로알려진 ligand 와결합하여활성화되는데, 활성화된세포는자신의 Notch ligand 를억제함으로써주위세포의 Notch 활성화를억제하게되며, 이와같은기전으로주위세포의분화결정에기여한다. 42 장관에서는 Notch 활성화는흡수장세포와분비세포사이의분화를결정하여, Notch 신호전달의억제는술잔세포의증식을유발하고, Notch 활성화는술잔세포결손을유발한다. 43,44 또한 WNT 신호와함께작용시세포의증식을촉진하고, WNT 신호없이단독으로작용할때흡수장세포로의분화를유도하여장선와항상성유지에관여한다. 43-45 대부분의대장암종양에서 WNT 신호가증가되어있으므로 Notch 신호에의한암줄기세포증식관련성이많이보고되어있고, Notch 신호조절에의한줄기세포의분화가잘알려져있으므로, 암줄기세포억제및분화치료에주요표적인자가될수있다. 3. BMP 및 Hh 신호와선와형성 BMP2 와 BMP4 는장점막의간엽세포에서발현되며, 선와-융모경계부위에서세포의증식억제와분화유도에관여한다. BMP 수용체결손또는 BMP 억제인자인 noggin 의과발현마우스는장선와의과증식소견을보인다. 46,47 BMP 는선와의상부뿐만아니라기저부에서도분비되지만장줄기세포부위의간엽세포에서분비되는 noggin 등의 BMP 억제인자는 BMP 의분화유도효과를억제하게된다. 상피유래의 platelet-derived growth factor (PDGF)-A 는융모형성과정에서융모중심부를형성하는간엽조직의증식을조절하는데관여하며, 특히 PDGF-A 자극에의해간엽세포로부터활성화된 BMP 신호는상피세포의증식구간내제한적증식에관여한다. 48 BMP 신호의이상은 familial juvenile polyposis syndrome (JPS) 과관련이있으며, BMP-4 억제단백질인 noggin 과발현마우스의경우 JPS 와유사한용종이발생하고, 47 BMP 수용체인 Bmpr1a 의불활성화역시동일한용종발생을유발한다. 46 이와같은 JPS 와유사한용종의발생은 BMP 신호억제에의해융모에서선와생성의비정상적활성화유발이관여한다고설명하고있다. BMP 신호활성화는분화유도와밀접한관련이있으므로암줄기세포의분화유도를통한항암제저항성억제에기여할수있는치료표적이다. Hh 신호전달역시배아발생의여러단계에서관여하며, 위장관발생에서 sonic hedgehog (SHH) 과 Indian hedgehog (IHH) 발현은상피줄기세포의증식과분화에관여하여, 발생기간동안 Hh 신호억제는선와-융모발생의이상을초래한다. 49-52 장선와의 Hh 신호전달에서, IHH 는분화된상피세포에서발현되어주위간엽세포에영향을주어 BMP 분비를유도한다. 52,53 SHH 의역할에대해서는아직이견이많으며그기능에대한자료가불충분하다. 이상의여러신호전달계의상호작용에의하여장선와의분화와증식이조절되며, 선와증식은점막손상의복구에도관여하여, 방사선조사에의한장점막손상후많은선와모양의구조를포함하는결절들이재생되는데, 54 이런재생선와들은작은분지들을형성하여새로운선와를형성하는선와생성주기를활성화시킨다. 이러한선와생성주기의활성화는점막손상복구뿐만아니라초기종양생성과정에서클론확장에도관여하여, 암줄기세포의발생과증식에중요한역할을한다. 최근이상과같은장선화항상성유지의기전에대한이해와함께장기간동안실험실배양조건에서장선와구조를유지, 증식할수있는방법이개발되었다. 55,56 WNT 관련인자, R-spondin-1, noggin, 그외성장인자등의조건에서장줄기세포의증식과장선와형성, 유지, 증식등을가능하게함으로써줄기세포의증식, 분화및 niche 의상호관계에대해배양상태에서의연구가가능하게되었다. 이와같은줄기세포연구방법의발전은암줄기세포의실험실내연구방법에서도적용되어유용한방법으로이용되고있다. 15,16 암줄기세포와미세환경의상호작용줄기세포의기능은장상피세포들의항상성을유지하는것으로자기복제, 증식, 그리고분화과정을통해장상피세포를일정하게유지한다. 이과정은 niche 라는주위구조내에서이루어지게되는데주위의간엽세포들과증식또는분화중인상피세포들에의해영향을받게된다. 비록기저막 (basement membrane) 에의해상피세포와분리되어있지만이곳의간엽세포 ( 세포외기질, 장신경계, 혈관, 상피내임파구, 선와주위섬유모세포 ) 들은줄기세포 niche 를유지하기위해필요한상피-간엽 (epithelial-mesenchymal) 간의상호작용을증진시킨다. 아직알려지지않은많은신호전달과분비인자가이런환경을유지하는데중요할것으로생각하며, 선와의바깥쪽을감싸는선와주위간엽 (pericryptal mesenchyme) 내의근섬유모세포 (myofibroblast) 는 HGF, transforming growth factor beta (TGFβ) 그리고 keratinocyte growth factor (KGF) 등의여러물질을분비한다. 3,57 그외선와 88 www.irjournal.org
http://dx.doi.org/10.5217/ir.2013.11.2.85 Intest Res 2013;11(2):85-91 주위간엽세포에서분비되어그에인접하여있는상피세포에영향을주는여러분비물질들로서 PDGF, SHH, BMP, forkhead-6 (Fkh), WNT 그리고 Notch 등이알려져있다. 장관선와내항상성유지를위해선와주위간엽조직은매우중요한역할을할것으로생각하며, 정상적인선와유지뿐만아니라암과같은병적상태에서도암세포와의상호관계를통해주요역할을한다. 최근침습성암종의진행에미세환경의미분화골수성세포가주요역할을함이보고되었고, 58 여러장염증모델에서의염증성매개체에의한암화과정촉진이알려져있다. 59 종양내염증세포들에의한 TNF-α, IL-6, TGF-β 등의성장인자및사이토카인들은종양의형성과증식에주요역할을하며, 60 상피세포의 NFκB 활성화를통해종양생성을증가시킬뿐아니라줄기세포가아닌암세포를암줄기세포로역분화시키는데에도기여한다. 19,61 이와더불어종양내섬유아세포도종양미세환경의주요세포로서여러종류의성장인자와사이토카인을분비하여종양의증식에관여한다. 62 특히종양내근섬유아세포에서분비되는 HGF 는 WNT 활성화를통해종양의증식과암줄기세포상태유지에관여하고, 또한 c-met 를통해분화된암세포를암줄기세포로역분화시키는데주요역할을한다. 18 이상과같이종양미세환경의여러세포군과분비인자들은종양의증식뿐만아니라암줄기세포의유지및증식에도관여하고있고, 특정미세환경조건에서는분화상태의암세포를암줄기세포의특성을지닌세포로변형시키기도하므로암줄기세포표적치료에서미세환경및 niche 인자의조절은빠질수없는표적이다. 18 결론대장암을포함한모든암질환의공통적인문제로서약제저항성, 재발, 전이의문제가암완치를위해해결해야할가장중요한문제일것이다. 이러한약제저항성, 재발, 전이의문제에공통적으로관여하는세포들의특성중하나가암줄기세포특성이므로암완치의꿈을위한여러연구들이암줄기세포를표적으로하여진행되고있다. 특히최근정상장줄기세포에대한연구의발전과함께정상줄기세포의유지및증식에서와유사한신호전달을이용하는암줄기세포에대한이해도많이발전하고있다. 그러나아직정상장줄기세포에대한영향을최소화할수있는암줄기세포특이적인표지자의발굴이쉽지않고, 암줄기세포의유지증식에관여하는영향인자로서암줄기세포의다양성, 미세환경인자및역분화관련인자등더욱복잡한상호관계가밝혀지면서암줄기세포에대한표적치료개발도많은어려움이예상된다. 그러나최근암줄기세포에대한분화치료개념및새로운관련인자규명등의중요한연구결과들과함께많은노력이집중되고있으므로암줄기세포를표적으로한치료제개발에한걸음가까워질것을기대한다. REFERENCES 1. Bjerknes M, Cheng H. The stem-cell zone of the small intestinal epithelium. I. Evidence from Paneth cells in the adult mouse. Am J Anat 1981;160:51-63. 2. Bjerknes M, Cheng H. Clonal analysis of mouse intestinal epithelial progenitors. Gastroenterology 1999;116:7-14. 3. Brittan M, Wright NA. Stem cell in gastrointestinal structure and neoplastic development. Gut 2004;53:899-910. 4. Barker N, van Es JH, Kuipers J, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007;449:1003-1007. 5. Breault DT, Min IM, Carlone DL, et al. Generation of mtert-gfp mice as a model to identify and study tissue progenitor cells. Proc Natl Acad Sci U S A 2008;105:10420-10425. 6. Montgomery RK, Carlone DL, Richmond CA, et al. Mouse telomerase reverse transcriptase (mtert) expression marks slowly cycling intestinal stem cells. Proc Natl Acad Sci U S A 2011;108:179-184. 7. Sangiorgi E, Capecchi MR. Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet 2008;40:915-920. 8. Lopez-Garcia C, Klein AM, Simons BD, Winton DJ. Intestinal stem cell replacement follows a pattern of neutral drift. Science 2010;330:822-825. 9. Snippert HJ, van der Flier LG, Sato T, et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 2010;143:134-144. 10. van der Flier LG, van Gijn ME, Hatzis P, et al. Transcription factor achaete scute-like 2 controls intestinal stem cell fate. Cell 2009;136:903-912. 11. Zhu L, Gibson P, Currle DS, et al. Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation. Nature 2009;457:603-607. 12. Huang EH, Hynes MJ, Zhang T, et al. Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res 2009;69:3382-3389. 13. Vaiopoulos AG, Kostakis ID, Koutsilieris M, Papavassiliou AG. Colorectal cancer stem cells. Stem Cells 2012;30:363-371. 14. Powell AE, Wang Y, Li Y, et al. The pan-erbb negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor. Cell 2012;149:146-158. 15. O Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007;445:106-110. 16. Ricci-Vitiani L, Lombardi DG, Pilozzi E, et al. Identification and expansion of human colon-cancer-initiating cells. Nature 2007;445:111-115. 17. Barker N, Ridgway RA, van Es JH, et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 2009;457:608-611. 18. Vermeulen L, De Sousa E Melo F, van der Heijden M, et al. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat Cell Biol 2010;12:468-476. 19. Schwitalla S, Fingerle AA, Cammareri P, et al. Intestinal tumorigenesis initiated by dedifferentiation and acquisition of stemcell-like properties. Cell 2013;152:25-38. 20. Dalerba P, Dylla SJ, Park IK, et al. Phenotypic characterization www.irjournal.org 89
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