Stem Cells and Cancer

1. Stem Cells and Cancer Donald Vander Griend, Ph.D. Assistant Professor Department of Surgery, Section of Urology

2. Today… • Pluripotency and Stem Cells • Tumor Initiating Cells (TICs) • Cancer Stem Cells (CSCs) • Stem Cell-Derived Cancers

3. Stem Cell Potency • Totipotent:can generate the whole organism including extra-embryonic tissues. Only the zygote and early blastomere are totipotent. • Pluripotent:can generate the whole organism but not the extra-embryonic tissues. ES cells are pluripotent. • Multipotent:can give rise to all cell types within one particular lineage. HSCs are multipotent. • Unipotent:only form one cell type. Spermatogonial stem cells are unipotent.

4. What defines Pluripotency? • Pluripotency describes a cell’s ability to give rise to all of the cells of an embryo and adult. • Recently, there has been an ongoing debate about what molecularly defines “pluripotency” of ES cells

5. Measuring Pluripotent Potential • In vitro differentiation:differentiation induced in cell culture and cells are assayed for the expression of cell-type specific markers. Limitation: marker expression is no test for functionality. • Teratoma formation:induction of tumors demonstrating the potential to generate differentiated cell lineages. Limitation: does not test within normal development, but most useful for human ES cells. • Chimera formation/Germline contribution:contribution of cells to normal development and generation of germ cells. Limitation: may not complement all cell types or over complement host defects. • Tetraploid complementation: injection of test cells into 4n blastocyst. Because 4n host cells cannot contribute to somatic lineages, embyro is exclusively composed of test cells.

6. Teratomas: Stem Cell-Derived Tumors • “Monstrous tumor” • Tumor tissue of embryonic origin that contains normal derivatives of all three germ layers. • Reported to contain hair, teeth, bone and even more complex organs such as eye, torso, hands, feet, and other limbs. Also may contain tissues resembling brain, thyroid, liver, and lung. • Can be derived from germ cells or embryonal cells.

7. Chimeric Mouse Pluripotent ES cells and Chimeras

8. Can Somatic Cells be Reprogrammed?

9. Takahashi and Yamanaka, Cell 2006 • “Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors.” • ES-specific promoter (Fbx15) driving a beta-gal/NeoR fusion gene (bgeo). Expressed combinations of cDNAs and screened for ES cell colonies.

10. Teratomas derived from Induced Pluripotent Stem Cells (iPSCs)

11. GFP IHC of Embryonic Tissues Chimeras derived from iPSCs (Derived from Adult Tail Tip Fibroblasts) GFP of Chimeras

12. WT mouse + Chimeric Mouse w/ Germline incorporation Okita et al. Nature 2007 Oct3/4 Sox2 Klf-4 c-Myc iPS Cells Liver Cells from Adult Mouse Control Generated F1 and F2 progeny from iPS cells.

13. Implications for Cancer • 7-10 day transient expression of these four factors caused an adult hepatocyte to become reprogrammed to a pluripotent ES cell. • Cells can definitely go backwards in their differentiation, including gene expression and methylation. • Such a phenotype does not require a stable genetic or epigenetic event. Can be a “hit and run” transformation event. • “Royal Flush” hypothesis

14. “Stemness” • Self-renewal: maintains a stable population of itself. • Hierarchy: generates phenotypically distinct progeny and lineages. • Durable: Enhanced mechanisms for survival and repair of damage. • Environment: Stem cells are highly regulated by their niche. • Asymmetric cell division (?)

15. Stem Cell Division and Generation of Lineages

16. The Immortal Strand Hypothesis Rando TA vs. Lansdorp PM; Cell 129, 2007

17. Stem Cells and Tissue Turnover(Epithelial Glandular Tissue) Epithelial Cell Type: Luminal/ Secretory Stem NE Basal Intermediate Renewal Capacity: Proliferation: Glandular Secretions: Terminal Differentiation

18. Differentiation: An Essential Concept in Stem Cell Biology Definition: The overall process of progenitor cells activating genetic and epigenetic mechanisms to define the specialized characteristics of mature cells. Characteristics: • Less or loss of proliferative ability • Loss of self-renewal capability • Moving towards tissue-specific biological function.

19. The Tumor Initiating Cell • Who Cares: Identifying the cell of origin for a type of cancer is a critical step in understanding carcinogenesis and disease prevention. • Cancers behave differently depending on the cell of origin. • Examples: • Breast Cancer:ER/PR/HER2 status. Triple-negative “basal-like” tumors are the worst. Also have Luminal A vs. Luminal B vs. HER2+ • Lung Cancer:NSCLC (squamous, adenocarcinoma, large cell carcinoma) vs. SCLC (neuroendocrine).

20. The significance of specific tumor suppressor gene mutations for determining the tumor initiating cell? • Loss of Rb predisposes to small cell lung carcinoma whereas p16 loss/silencing predisposes to non-small cell carcinoma • Does this reflect differential roles of these genes in the respective stem cells of the lung, ie. neuroendocrine versus epithelial?

21. Identifying the “Tumor Initiating” Cell. • Tumors are clonally derived from a single aberrant cell, the “tumor initiating cell”. • Tissue stem cells are attractive candidate “tumor-initiating” cells since they share with tumor cells the capacity for self-renewal and clonal expansion. • Do tumorigenic mutations need to occur in stem cells in order to propagate the tumor on a long term basis? • The turnover of cycling progenitors may be too rapid to allow them to accumulate all the required mutations to develop into a tumor”. • Stem cells not subject to selection until they expand allowing them to acquire mutations while “resting”. Mutations in oncogenes/TSGs may not have severe phenotypic consequences for the non-proliferating stem cell pool but would be tumorigenic in the highly proliferative daughter cells. • Are stem cells therefore a reservoir of oncogenic mutations? • If stem cells are the tumor-initiating cells for most cancers then this may explain why therapies that target proliferating cells (tumor cells) fail to eliminate cancer on a long term basis since the mutant stem cell pool would survive and act as a reservoir of potential tumor cells.

22. Limitless self-renewal properties Quiescent Limited self-renewal properties Highly proliferative TUMOR CELLS Limitless self-renewal properties Highly proliferative Do tumor arise clonally from “immortal” stem cells that acquire proliferative potential, or from highly proliferative progenitors that acquire self-renewal capacity?

23. Stem cells acquire proliferative/survival potential and their expanded progenitor progeny then acquire self-renewal potential.

24. Cancer Stem Cells: Not A New Concept • The concept that cancer might arise from a rare population of cells with stem cell properties was proposed about 150 years ago (German Papers). • Over 40 years ago it was postulated that tissue-specific stem cells may be the cell of origin of cancer (Till et al., 1961). • In 1971, Pierce et al. showed experimentally that cellular hierarchy existed in squamous cell carcinomas in vivo. They found that malignant undifferentiated cells gave rise to benign well-differentiated cells. • Potter, in 1978, championed the concept of “oncology is blocked ontogeny” suggesting that a lack of differentiation plays a critical role in cancer.

25. Identifying Cancer Stem Cells • Controversial “Stem” of Caution: the use of immunodeficient mice for transplantation may not accurately reflect what occurs in human cancers. • Nude • SCID • NOD-SCID • NOG-SCID

26. Cancer Stem Cells vs. Clonal Evolution Cancer Stem Cell Model Rare to Moderate Heterogeneous Hierarchical Stable, epigenetic Possible to target only tumorigenic cells Germ lineage tumors Stochastic Clonal Evolution Model High Heterogeneous or Homogeneous Not hierarchical Unstable, epigenetic or genetic Target most or all cells HG B Cell lymphoblastic leukemia • Frequency of cancer cells with tumorigenic potential • Phenotype of cancer cells • Tumor organization • Intrinsic differences between tumorigenic and non-tumorigenic cells • Rational approach to therapy • Compelling clinical evidence

27. High Frequency of Cancer Forming Ability (Stochastic Model) Kelly et al.; Science 2007 Quintana et al.; Nature 2008

28. A Cancer Stem Cell ExampleO’Brien et al.; Nature 2007 • CD133+ populations from patients with colon cancer, injected as xenografts into NOD-SCID mice • Questions: • CD133+ cells more tumorigenic that CD133- cells (initiating cells)? • Tumor xenograft from CD133+ cells resemble original tumor (subset)? • CD133+ derived tumors contain CD133- populations (lineages)? • Do CD133+ cells from tumors able to re-derive more tumors (self-renewal)?

29. Clinical Relevance of CSCs

30. Cancer Stem Cell Hypothesis Heterogeneous Tumor More effective gene specific therapies will cure. Treatment Relapse Cancer Stem Cell Model More effective anti-tumor therapies will reduce but NOT cure. Pursue different target. * Relapse

31. Multiple Myeloma CSCs Exhibit Differential Sensitivity to Drugs

32. Other Clinical Considerations • Markers for early detection: detection of CSC populations vs. differentiated cells. • Cancer Prevention: in some instances normal stem cell expansion is the first event in carcinogenesis (inflammation). • Clinical Trials: short term vs. long term outcomes (survival). An agent that kills CSCs may have no measureable short-term benefit.

33. Identifying Stem-Like Populations

34. Surface Markers Sphere Formation

35. ABCG2: ATP-Binding Cassette Transporter Type G2 • Also called BCRP (Breast Cancer Resistance Protein) • Originally identified as being over-expressed in a Multiple Drug Resistant (MDR) breast cancer cell line. • Implicated in the side population phenotype for stem cells (SP). P-Glycoprotein (ABCB1) BCRP (ABCG2) - Homodimerizes Out Out Figure from A.H. Schinkel, J.W. Jonker / Advanced Drug Delivery Reviews 55 (2003) 3–29

36. Side Population to Identify ABCG2/BCRP+ Cells

37. ALDH: Aldehyde Dehydrogenase • 12 ALDH genes in humans • Oxidize a variety of aliphatic and aromatic aldehydes. • General detoxifying enzymes • ALDH1 is a cytosolic enzyme which is important in retinoic acid signaling. Figure from Aldagen Talk; Michelle Fiordalisi, Ph.D.

38. ALDEFLUOR: Overview ALDH1 Substrate (weakly fluorescent) Product (strongly fluorescent) DEAB Cells ALDH1+ ALDH1+ Bright + ALDH1- ALDH1- Dim

39. ALDEFLUOR to Detect ALDH1 Activity

40. CD133 • Wider expression in embryonic tissues • Preference for plasma membrane protrusions • A.K.A. Prominin, AC133 • Human - 4p16.2-p12 • Location: Stem and Progenitor Cells, photoreceptor cells (Figure from Corbeil et al. Traffic 2001 2: 82–91)

41. QUESTIONS? prostate@uchicago.edu