cancer stem cells - overview

1. CANCER STEM CELLS
DR. NEELAM AHIRWAR
DNBSS RESIDENT(SURGICAL ONCOLOGY)
APOLLO CBCC,AHMEDABAD

2. Cancer stem cells (CSCs)
• Introduction
• Origin
• Niche
• EMT
• Regulation
• Resistance to therapy and Recurrence
• CSC and metastasis
• Identification and isolation techniques
• Therapeutics

3. Introduction
• Cancer stem cell term was coined by Sajiro Makino
• first identified in AML, a population of CD34+CD38− stem cells
capable of initiating AML in severe combined immune-deficient
(SCID) mice.
• recognised in a number of solid tumors, including breast cancer,
brain tumors, lung cancer, colon cancer, and melanoma.

4. Introduction (CSCs)
• A small subpopulation of cells within a tumor
• Express cell surface markers including CD44, CD24 and/or CD133.
• Stemness - capacity to self-renew and to generate differentiated cells that
contribute to tumour heterogeneity
• Capable of regenerating a tumor when transplanted into an animal host.
• Epithelial mesenchymal transition (EMT) may either enhance or inhibit cscs
in a cell type specific manner.

5. Introduction (CSCs)
• miRNAs are involved in the regulation of properties
• Resistant to conventional treatment (chemo and radiation)
• Important roles in cancer relapse and metastasis.

6. Origin of CSC- Hypothesis
• Stochastic model –
• any cell subjected to a specific set of somatic mutations
• acquire self-renewal and differentiation properties
• contribute to tumor heterogeneity
• Hierarchical model
• somatic mutations target specifically stem & progenitor cells
• generation of self-renewing cancer stem and progenitor cells
• produce differentiated progenies and recreate heterogeneous tumors

7. Niche of cancer stem cells
• Complex microenvironment
• Cancer associated fibroblasts (CAFs)
Endothelial cells
Mesenchymal stem cells (MSCs)

8. Niche of cancer stem cells
• communicate through secreted growth factors and cytokines
• promote tumorigenesis, angiogenesis, invasion and metastasis.
• promote the growth, maintenance and differentiation of cancer stem
cells
• contributes to CSCs’ resistance to drugs and

9. Epithelial To Mesenchymal Transition
EMT refers to cellular process when an epithelial cell loses adhesion and
adopts a mesenchymal morphology.
EMT is controlled by Signaling pathway
Environmental factors
Transcriptional factors(snail1,twist1,zeb1)
Micro RNA

10. Signaling pathway

11. Epithelial To Mesenchymal Transition

12. EMT- 1. TGF β signaling
TGF-β binding to
its receptors
activation of the SMAD proteins
activate target genes involved in EMT (SNAIL, ZEB
factors)
promote the expression of mesenchymal proteins such as N-
cadherin, fibronectin and metalloproteinases (MMPs)

13. EMT
2.WNT signaling - stabilising β-catenin through the inhibition of glycogen
synthase kinase-3β (GSK3β), resulting in β-catenin translocation to the nucleus
and activation of the transcription factors lymphoid enhancer-binding factor 1
(LEF) and T cell factor (TCF), thus promoting a gene expression programme that
favors EMT
3.SHH signaling increases SNAIL1 expression in epithelial cancers leading to
increased EMT and invasion

14. EMT
4.Notch signalling may promote EMT through the Notch intracellular
domain that directly activates SNAIL2 expression.

15. EMT
• Multiple Myeloma have displayed abnormal signaling in response to
elevated levels of Hedgehog ligand secreted by tumor stromal cells
• upregulated Notch4 signaling – drug resistant Breast CSC activity
• Invasive breast carcinoma -overexpression of Snail genes correlating
with activation of Akt and b-catenin pathways
• Cellular co-expression of Snail, Slug and E-cadherin - breast and colon
carcinoma cells

16. EXTRINSIC REGULATION OF CANCER STEM CELLS
HYPOXIA
• Rapid proliferation and aberrant angiogenesis lead to hypoxia regions
within tumors.
• Knockdown of HIF1a and HIF2a decreases self renewal and tumor
propagation of CD133+ Brain CSCs.
• In CML ,HIF1a in bone marrow protects leukemic SCs from Imatinib
therapy.

17. EXTRINSIC REGULATION OF CANCER STEM CELLS
INFLAMMATION
• regulates tumor initiation ,progression, and CSCs properties
• stimulates EMT.
• Colorectal cancer cells exposed to IL6 repress miR34,which allows
expression of EMT , invasion and metastasis.
• Her 2+ breast cancer cells resistant to Transtuzumab therapy are enriched
in CSCs and show EMT features as well as major upregulation of IL6.

18. EPIGENETIC REGULATION
• CSCs regulated by Histone modifications and DNA methylation.
• Histone acetylation regulates transcription activation in cancer cells
• Histone deacetylase inhibitors
-target leukemia Sc
-increase radiosensitivity of prostate cancer
-sensitize SCC of head and neck to treatment by Gefitinib.

19. Cancer therapy resistance and recurrence

20. Cancer therapy resistance and recurrence
Mechanisms that counteract the effect of chemotherapeutic agents
• ATP family of transporters
• Higher expression of the pro-survival factor BCL2 (b-cell lymphoma 2)
• Higher expression of aldehyde dehydrogenases (ALDH),
• More efficient DNA damage response
• Slower rate of cell division or dormancy

22. Expression of ATP-Binding Cassette (ABC) Transporters
• Transport of chemotherapeutic compounds out of the cells
• In a glioblastoma model, aberrant Akt signaling contributed to over
activation of the ABC transporter ABCG2 in CSCs, leading to increased
drug expulsion and rendering them resistant to mitoxantrone

23. Increased Expression of Aldehyde Dehydrogenases (ALDHs)
• Crucial for the detoxification of endogenous and exogenous aldehyde
substrates
• Catalyze the oxidation of aldehydes to carboxylic acids
• Elevated levels of expression correlate with worse prognosis in cancer
patients
• Breast cancer -stem cells are identified using specific antibodies for CD44
and CD24 and an ADELFLUOR assay to detect ALDH activity.
CD44+/CD24−/ALDH+ populations have been shown to possess
characteristics of stem cells

24. Resistance to Apoptosis
• Resist apoptosis- the intrinsic and extrinsic pathway
• Human Glioma Cells and leukemia CSCs express lower levels of Fas
and Fas ligand (Fas-L) resulting in resistance to extrinsic receptor-
mediated cell death
• The pro-survival protein Bcl-2 (intrinsic pathway) via its inhibitory
interactions with the proapoptotic Bax (Bcl-2-associated x) and Bak
(Bcl-2 antagonist killer), was found overexpressed in leukemia, glioma
and glioblastoma stem cells.

25. Low Proliferation Rate
• Chemotherapy targets highly proliferative cells
• CSC from different tissue origin resist the effect of a wide range of
drugs such as doxorubicin, temozolomide, cisplatin, paclitaxel.
• Following chemotherapy cscs remain on-site and can re-initiate
tumorigenic and metastatic processes.

26. CANCER STEM CELL AND METASTASIS.
• Both metastatic dissemination and the acquisition of CSCs promoted
by EMT.
• Additional features such as plasticity and dormancy.
• Metastasis can occur years after successful treatment ,and during this
period CSCs remain quiescent.

27. CANCER STEM CELL AND METASTASIS
Mammary tumors (CD24+ CD90+ CSCs)
Induces expression of stromal periostin at metastatic site and their
expansion
Metastasis
Blocking function of periostin prevents metastasis.

28. CSC- Identification and isolation
• Flow Cytometric Analysis Of Csc-specific Cell Surface Markers
• Detection Of Side-population (SP) Phenotypes By Hoechst 33342 Dye Exclusion
• Aldeflour Assay (Changes In ALDH Enzymatic Activities)
• Ability To Grow As Suspension Spheres In Serum-free Medium,
• SC related Gene Expression
• Auto-fluorescence.

29. CSC- Identification and isolation

30. CSC- Identification and isolation
• Surface Marker-based Assays by Fluorescence-activated Cell Sorting
Method - most commonly used

31. Therapeutic targeting against CSCs

32. 1.Targeting cell surface receptors
• CD44 antibody therapy (H90)as the major anti-CSC successfully
eradicated AML
• CD133 is a CSC marker in glioblastoma, hepatocellular and colon
cancers. AC133 antibody was conjugated to a potent cytotoxic drug,
monomethyl auristatin, using a protease cleavable linker. This
antibody drug conjugate showed high effectiveness against
hepatocellular cancer Cells

33. 1.Targeting cell surface receptors
• Humanized EpCAM antibodies show potent anti-tumor activity in
breast cancer
• Adecatumumab, a recombinant human IgG1 monoclonal Ab was
clinically tested in patients with metastatic breast cancer

34. 2. Targetting the signaling pathway
• Psoralidin inhibited Notch signaling in both bulk tumor and CSCs
-decreased mammosphere formation,
-upregulationof pro-apoptotic proteins
-inhibition of CSC proliferation in breast cancer
• Cyclopamine was the first Hedgehog antagonist to be identified
-induced tumor regression in a CML model
-decreased tumor growth rate in a medulloblastoma model
-inhibited proliferation of pancreatic CSCs.

35. 3. Targetting the Microenvironment
Repertaxin, a non-competitive inhibitor of IL-8 and CXCR1 signaling,
decreased tumor size and increased efficacy of chemotherapy.
co-inhibition of both IL-6 and IL-8 was suggested to be a more
advantageous.
Bortezomib (Velcade , PS-341) was approved by the FDA in 2003 for use
against multiple myeloma

36. 3.Targetting the Microenvironment
• Temsirolimus (Torisel , CCI-779), approved in 2007 for use against renal cell
carcinoma
• Bevacizumab (Avastin ), Cediranib (AZD2171), Sunitinib and Vandetanib- VEGF
inhibitors (prevents angiogenesis)
• In bladder cancer CT increases level of PGE2 which induces proliferation of
dormant CSCs. Blockage of PGE2 with cox2 inhibitors abrogated this response

38. Nanocarriers
• enhanced the delivery and cytotoxic activity of CSC-inhibitors
• High target selectivity and internalization can be achieved by surface
modification of nanoparticles with targeting moieties
• targeted nanoparticles induced a significant tumor volume reduction
compared in an in vivo MDA-MB 231 xenograft tumor model by developing
paclitaxel-loaded polymeric PLGA nanoparticles conjugated with CD133
mAb.
• In myeloma CSCs were treated with silver nanoparticles decorated with
anti-ABCG2 antibodies on the surface along with vincristine.

39. Summary
• the specificity of the targets is key for a successful eradication of CSCs
and cancer recurrence
• important to identifiy CSC-specific genes or antigen signatures
• development of more efficient CSC targeted therapies
• lower associated toxicities for treated patients.

40. Thank you