Stem Cells in Cancer: A Review

Jovana J. GrbićSchultz Group Seminar

October 18, 2005

Stem Cells in Cancer: A Review

• Introduction and Background

– What is a stem cell? What is a cancer stem cell?– Epithelial cancer stem cell characteristics– How many insults could you take?– How do you know it’s the stem cells?– Therapeutic Implications– Where no scientist has gone before….

• Specific Case Studies:– Colorectal Cancer (CRC) and epithelial stem

cell renewal– Breast Cancer: mammary stem cells– AML/CML leukemias: leukemic stem cells

What is a Stem Cell? What is a Cancer Stem Cell?

• Fundamental homeostatic system for replacing terminally differentiated cells of the body due to senescence and injury

• Involves stages of division with decreasing reproductive capacity and increased commitment to differentiation

• Self-replication and progeny production

• Cancerous growth is abberant normal tissue homeostasis

Stem cell→committed progenitor→diff. cell

• Tumor stem cell model mimics normal tissue models

• Variations in tumor growth rates: variations in homeostatic mechanisms of SC regulation and preturbations in the niche microenvironment

• CSC’s arise from a multi-step process that parallels the slow development of cancer from normal tissues

Biochim Biophys Acta. 2005 Sep 25;1756(1):25-52

Nat Rev Cancer. 2005 Apr;5(4):275-84

How Many Insults Could You Take???

• I: Pretumor Progression– Mutational acquisition

(prenatal)– Additional mutations– Clonal expansion of stem cell

“niche”

• II: Patches– CSC ventures outside its niche– Present in phenotypically

normal epithelia

• III: Fields– Larger regions of clonal

outgrowth, may begin to manifest histologic atypia

Therapeutic Implications

• Multi-Drug Resistant Proteins– ABCG2 inhibitors, ABCG2

antibodies, stem cell inhibitors, immunotherapy

• Irradiation– Stem cells may be more

sensitive to irradiation

• Differentiation, apoptosis, and senscence

Where No Scientist Has Gone Before

• Identification of CSCs– Similar phenotype, slow cycling, constitute small percentage, BUT contain

many unique markers as well.

• Why can’t ablate with traditional chemotherapy?– Target rapidly dividing cells, overexpress a lot of ABC drug-transporters

• How do early epithelial CSCs migrate?– Form temporary migratory phenotype (i.e. wound healing, patches/fields)

• How are CSCs removed from a tumor population?– No mechanism as of yet for total progression from CSC to committed cell

lineage with loss of stem cell population

• Different cancers in the same organ:– Different malignant stem cells or different “packaging” of the same stem

cell?

Stem Cells in Cancer: A Review

• Introduction and Background

– What is a stem cell? What is a cancer stem cell?– Epithelial cancer stem cell characteristics– How many insults could you take?– How do you know it’s the stem cells?– Therapeutic Implications– Where no scientist has gone before….

• Specific Case Studies:– Colorectal Cancer (CRC) and epithelial stem cell

renewal– Breast Cancer: mammary stem cells– AML/CML leukemias: leukemic stem cells

Endothelial Gut Lining: Physiology and Stem Cell Renewal

Science. 2005 Mar 25;307(5717):1904-9

Colon epithelium

Canonical Wnt Signaling Maintains Crypt Progenitor Compartments

Cell. 2002 Oct 18;111(2):251-63Nat Genet. 1998 Aug;19(4):379-83

Wnt cascade is dominant force in controlling cell fate along crypt-villus axis:

~Staining for nuclear β-catenin mainly observed in crypts

~Tcf4 -/- mice lack proliferative compartments for maintaing progenitors

Intestinal Tumorigenesis:Mutational Wnt Pathway Activation

• Most molecular insights of CRC come from hereditary studies (5-10% of cases)

• Familial adenomatous polyposis (FAP): patients carry numerous polyps, invariably develop CRC around age 40

• Carry one mutant copy of APC– C-terminal truncation

• Inappropriate activation of intestinal TCF4—imposes proliferative phenotype in crypt epithelial cells

Cell. 2002 Oct 18;111(2):241-50

Science. 1997 Mar 21;275(5307):1784-7

Notch/bHLH Signaling

Science. 2005 Mar 25;307(5717):1904-9

• Important for regulating cell fate decisions/differentiation

• Most likely target is Hes family of bHLH proteins– Notch target genes control

intestinal homeostasis

• Hes1-/- mice contain increased secretory cells at the expense of absorptive enterocytes

Nat Genet. 2000 Jan;24(1):36-44.

CRC: Conclusions

• Crypt-villus unit represents one of the simplest self-renewing entities in mammalian biology

• Regulatory signaling pathways have emerged from embryonic development studies as a key in maintenance of this structure

• Transformation process probably manipulates key physiological regulators of normal intestinal epithelium as most efficial roadblocks towards cancer (not mutational changes in generic oncogenes or tumor suppressors)

Stem Cells in Cancer: A Review

• Introduction and Background

– What is a stem cell? What is a cancer stem cell?– Epithelial cancer stem cell characteristics– How many insults could you take?– How do you know it’s the stem cells?– Therapeutic Implications– Where no scientist has gone before….

• Specific Case Studies:– Colorectal Cancer (CRC) and epithelial stem cell

renewal– Breast Cancer: mammary stem cells– AML/CML leukemias: leukemic stem cells

• Most common malignant disease in Western women

• Despite advances, metastatic breast cancer remains a death sentence– Current treatment is limited by

emergence of therapy-resistant cells

– Early detection/treatment have drastically cut overall death rates

• Adequate predictions of metastatic invasion are KEY– 80% of women receive adjuvant

chemotherapy, only 40% need it

J Cell Sci. 2005 Aug 15;118(Pt 16):3585-94

Breast Cancer Background InfoMammary Arbor Architecture and

Cell Engineering

•Label-retention studies: 5-Br2-dU

•Cell-surface markers (Sca-1, CD49f)

•DNA-binding dyes: Hoechst efflux

•Hormone Receptor Status (ER, PR)

Signalling Pathways and Mammary Stem Cell Regulation

• Originally identified as a viral insertion in a MMTV-induced mammary tumor

• Stabilization of β-catenin in >50% breast cancer tumors (poor prognosis)

• Knockout mice lacking LEF/mice expressing Dickkopf (Wnt inhibitor) fail to develop mammary glands

• Wnt signaling plays a role in several human ovarian/breast cancer cell lines

• Important role in neoplastc mammary stem-cell-like progenitors– Expansion of Sca1+ progenitors in Wnt+

transgenic mice

– Did not observe such expansion in non-Wnt mediated tumor models

Wnt

Cancer Cell. 2004 Nov;6(5):497-506

Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15853-8

Signalling Pathways and Mammary Stem Cell Regulation

• Originally identified as a viral insertion in a MMTV-induced mammary tumor

• Stabilization of β-catenin in >50% breast cancer tumors (poor prognosis)

• Knockout mice lacking LEF/mice expressing Dickkopf (Wnt inhibitor) fail to develop mammary glands

• Wnt signaling plays a role in several human ovarian/breast cancer cell lines

• Important role in neoplastc mammary stem-cell-like progenitors– Expansion of Sca1+ progenitors in Wnt+

transgenic mice

– Did not observe such expansion in non-Wnt mediated tumor models

Wnt

Cancer Cell. 2004 Nov;6(5):497-506

Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15853-8 Keratin-6 staining

Signalling Pathways and Mammary Stem Cell Regulation-II

• Important in normal mammary development and identified as MMTV insertion site in mammary tumors

• Notch4 transgenic mice develop mammary tumors

• Role of Notch in formation of mammospheres:

• Important for normal development of virtually every organ system

• Pathway components shown to be OE in many cancer types (including breast)

• Majority of human breast cancers show uniform OE of PTCH1 and nuclear Gli1

• Cyclopamine confers growth inhibition of a subset of breast cancer cell lines in vivo

Notch Hedgehog

Breast Cancer Res. 2004;6(6):R605-15. Cancer Res. 2004 Sep 1;64(17):6071-4.

Identification and Isolation of Tumorigenic Breast Cancer Cells

PNAS U S A. 2003 Apr 1;100(7):3983-8

• Exponential increase in breast cancer with woman’s age

• Stem cells are an attractive target due to unlimited replicative potential

• Cell-surface markers used to identify/isolate subpopulation of tumorigenic cells– CD44+/CD24- cells show increased tumor formation

– As few as 100 cells can recapitulate human tumors

– CD44+/CD24- cells can be cultured indefinitely

Mammary Stem Cell Progression Model

CANCER!!!NORMAL TISSUE

Conclusion: Clinical ImplicationsProposed Future Breast

Cancer TreatmentKaplan-Meier Survival PlotBased on CD44 Expression

Histopathology. 2003 Jun;42(6):546-54

CD44+ breast tumor cells confer both lower rates of overall and disease-free survival

LESSONS FROM CURRENT TREATMENTS:

-Tumors can undergo accelerated repopulation between daily radiation doses (clonogenic assays)

-Tumor stem cells likely more resistant to both chemotherapy/radiation (repopulate from added stress of treatment)

-Treatment of Sca1+ mammary cells with taxol or radiation leads to increase in clonogens formed in matrigel

Stem Cells in Cancer: A Review

• Introduction and Background

– What is a stem cell? What is a cancer stem cell?– Epithelial cancer stem cell characteristics– How many insults could you take?– How do you know it’s the stem cells?– Therapeutic Implications– Where no scientist has gone before….

• Specific Case Studies:– Colorectal Cancer (CRC) and epithelial stem cell

renewal– Breast Cancer: mammary stem cells– AML/CML leukemias: leukemic stem cells

Hematopoiesis

Nat Rev Immunol. 2005 Jun;5(6):497-508

Hematopoietic system constantly generates specialized cell types from HSC progenitors:

The stem cell niche supports HSC regeneration/maintenance via stromal cells and osteoblasts

Earliest bifurcation is the multi-potent progenitor, which branches off into the common myeloid progenitor (E, M, MK, G) or common lymphoid progenitor

Lymphoid progenitors can branch into the pro-T-Cell or pro-B-Cell differentiation pathways depending on gene expression

Blood. 2005 Apr 1;105(7):2631-9.

Acute Myelogenous Leukemia Models

Nat Rev Cancer. 2005 Apr;5(4):311-21

Leukemias are myeloproliferative disorders that are clonal in nature

Earliest identified model for CSCs (LSC): only 1/1,000-5,000 cells could form colonies…massive cell injections in mice to replicate leukemias

LSCs express similar surface markers (CD34+/CD38-), telomerase levels, and are able to repopulate diverse cancer progenitors in mouse models

Stem cell pathway candidates for LSC transformation

NOD/SCID Mouse expansion of normal and neoplastic HSCs

Alternative HSC transformation model

Leuk Res. 2005 Sep 21 Epub

Polycomb group gene Bmi-1 determines the proliferative capacity of normal and leukemic stem cells

•Indispensable for generating AML in mice•Only 1% of Bmi-1 -/- cells could form colonies (>1010 cells) while 24% of Bmi-1+/+ cells did•Bmi-1 -/- weakly leukemogenic in vivo (transplants), but the effect is rescued by forced expression of Bmi-1 in these cells•From this data, we may construct more sophisticated models for LSC generation (i.e. cooperating genetic events, TLS-ERG)

Nature. 2003 May 15;423(6937):255-60.

Leukemia. 2005 Oct;19(10):1794-805

Chronic Myelogenous Leukemia

Nat Rev Cancer. 2005 Mar;5(3):172-83.

The Philadelphia Chromosome: t(9;22)(q34;q11)

Largely viewed as biphasic: Bcr-Abl translocation/expression in HSCs followed by expansion of CML-chronic phase granulocytes

Additional mutations (many discussed today) lead to blast phase CML: uncontrolled expansion of either myeloid (2/3) or lymphoid (1/3) blast cells

Gleevec

Therapeutic Directions

Nature. 2005 Jun 30;435(7046):1169-70

Nat Rev Cancer. 2005 Apr;5(4):311-21

Mathematical model of CML reoccurence post-Gleevec treatment:

S= LSC

P= Leukemic progenitors

D=differentiated LS

TD=terminally differentiated

Nature. 2005 Jun 30;435(7046):1267-70

Leukemic stem cells are NOT depleted by Gleevectreatment

Future cocktail therapies must address this problem