Stem Cell Technology – Unit 5

15/11/2024
Why does a tumor not respond to treatment?

Why tumors recur?

Why cancer cells develop resistance to

treatment?

At present, the shrinkage in the size of a tumor is

considered as a response to the treatment.
However, tumor often shrinks in response to the
treatment only to recur.
These and many other raised questions may be
answered by the new concept of
"Cancer Stem Cells”.
Hierarchy of stem cells
Normal stem cells are characterized by three
properties:

1 Capability of self-renewal;
2 Strict control on stem cell numbers;
3 Ability to divide and differentiate to generate
all functional elements of that tissue
In normal tissue, three different compartments
can be described:

 a self-renewal compartment with quiescent

stem cells,
 a proliferating compartment with proliferating
progenitors with a limited potential for self-
renewal and
 a terminal compartment with differentiated
cells or apoptotic cells.
Cancer:
 Failure in the mechanisms that usually
control the growth and proliferation of cells.
 Cancer commonly results from mutations
that arise during a lifetime exposure to
carcinogens like UV or certain chemicals.
 Cancer forming process called
oncogenesis/tumorigenesis is an interplay
between genetics and the environment.
types of cancer

- Benign
- Malignant
Causes of certain cancers are obvious:

1. smoking-lung cancer, oral cancer

2. exposure to UV radiation-skin cancer

Several viruses infect mammalian cells,

causing transformed cells:

- DNA tumor virus (Polyoma virus, Simian Virus 40,

adenovirus)

- RNA tumor virus (retrovirus example; HIV)

- Most of the viruses accelerate than inducing
cancer.

- Chronic inflammation along with pathogen or

infection may cause cancer.
- Human Papilloma Virus (HPV) – sexually
transmitted - cervical cancer and 90% not
malignant

- Other virus related to human cancer:

Hepatitis B – liver cancer
Sequences of events associated in the
progression of cancer
Mutation
(i) Somatic mutation
(ii) Germline mutation

Genetic instability

Selection

Clone of altered cells -TUMOR INITIATION

TUMOR PROGRESSION-
Metastasis
Angiogenesis
Evasion of apoptosis

CANCER
Breast Cancer Progression
 Bone Metastases

Histological Appearance
Osteolytic Bone Metastases
of Metastatic Tumor Cells

Selvamurugan et al.
 Genetic Basis of cancer

Mutation occurs in two broad classes of genes

(i) Proto-oncogenes – activation causes
cancer
(ii) Tumor suppressor genes – deactivation
causes cancer
 Oncogene vs Tumor Suppressor Gene
A tumor suppressor gene is defined as
An oncogene is defined as a mutated a normal gene that is found in the cells
proto-oncogene that has the potential to of the body that could be transformed
develop cancer. into a tumor causing gene due to a
mutation.
Relation to Cancer
Tumor suppressor gene protects cells
Oncogene causes a cancer.
becoming cancerous.
State of the Gene When Causing Cancer
Tumor suppressor genes are causing
Oncogenes are in the active form when
cancers when they are in the inactive
causing cancers.
form.
Mutation Occurrence
The tumor suppressor gene mutation
Oncogene mutation occurs in the
occurs in both germ cells and somatic
somatic cells.
cells.
Inheritance
If the tumor suppressor gene mutation
Since the oncogene mutation occurs in
occurs in the germ line cells, it has the
somatic cells, it is not inherited.
potential to be inherited.

Pharamaceutical Microbiology, 2018

Behavior of Cancer Cell
 Cancer Stem Cells Identification:

When cancer cells of different origins were

analyzed for their proliferative potential in
various in vitro and in vivo assays, only a small
minority of cells were able to proliferate
extensively.
A simplified model of suggested hypothesis
about origin of the cancer stem cells
Cancer stem cells are derived either from
transformed normal stem cells or from more
differentiated progenitor cells that have acquired
the ability to self-renew because of oncogenic
mutations.
 Cancer Stem Cell Hypothesis

 Not all the cells in the tumor can proliferate and

maintain growth of the tumor, but only a small
subpopulation of cells in the tumor, called cancer
stem cells, are able to proliferate and self-renew.

 Cancer stem cells have been identified in

leukemia, breast, lung, and brain cancers.
Breast Cancer Stem Cells
 Breast Cancer Stem Cells:

 Cancer stem cells could potentially be

derived from bipotential stem cells or from
more differentiated cells that acquired self-
renewal capabilities.

 Cancer stem cells have restricted

differentiation potential.

 Produce tumors with features of a

particular lineage (luminal or basal like).
 Cancer Therapy

How to increase the efficacy of cancer therapy:

by eliminating cancer stem cells.
For tumors in which the cancer stem cells
play role, three possibilities exist.
 First, the mutation of normal stem cells or progenitor
cells into cancer stem cells can lead to the development
of the primary tumor.

Second, during chemotherapy, most of the primary

tumor cells may be destroyed but if cancer stem cells
are not eradicated, they become refractory cancer stem
cells and may lead to recurrence of tumor.

Third, the cancer stem cells may emigrate to distal sites

from the primary tumor and cause metastasis.
The conventional therapies may shrink the size of the
tumor; by contrast, if the therapies are directed against
the cancer stem cells, they are more effective in
eradicating the tumor.
Theoretically, identification of the cancer stem
cells may allow the development of treatment
modalities that target the cancer stem cells
rather than rapidly dividing cells in the cancer.
 Why cancer stem cells are resistant to
chemotherapy?

 cancer stem cells are quiescent

 have high levels of ABC transporters (MDR-expel
the drug from cell)
 have high capacity for DNA repair and
 express high levels of anti-apoptotic proteins.

ATP-binding cassette transporters

Elimination of cancer stem cells:

 transplantation of allogeneic hematopoietic stem cells

with graft versus leukemia/tumor effect,

 targeted therapies against self-renewal signaling

pathways and the cell cycle,

 modulation of chemoresistance mechanisms or

tackling other specific properties of cancer stem cells.
 Several molecular signalling pathways
including PTEN, the polycomb gene, Wnt, Hh
and Notch play a role in normal stem cell self-
renewal.

 It is possible to selectively target these

pathways involved in normal and cancer stem
cell self-renewal.

 Recent studies showed that cancer stem cells

and normal stem cells share the Bmi1 and Wnt
pathways, i.e. mechanisms for self-renewal.
 Furthermore, understanding the molecular
mechanisms of cell cycle control in stem cells
could also provide new strategies for cancer
stem cell therapies.

 One way to handle the cancer stem cell (CSC)

problem is to treat cancer by inducing
differentiation of cancer stem cells.

 Differentiation therapy could force cancer

stem cells to differentiate and lose their self-
renewal property.
 Vitamin A and its analogues (Retinoids) are
modulators of differentiation and proliferation of
epithelial cells.

 They can invert the malignant progression

process through transduction signal modulation
mediated by nuclear retinoid receptors.

 HDAC inhibitor, suberoylanilide hydroxamic

acid (SAHA) can induce differentiation of
cultured murine erythroleukaemia cells.
Identification of new and future therapies:

 Differences between normal and cancer stem

cells could involve transcriptional, post-
transcriptional, translational and post-
translational modifications and that could
identify novel diagnostic markers and novel
therapeutic targets.

 The cell surface marker which is differentially

expressed between normal and cancer stem
cells could be a new target for antibody-based
therapy.
 Cancer stem cell pathways

 The design of new drugs for the treatment of CSCs will

likely require an understanding of the cellular
mechanisms that regulate cell proliferation.

 A normal stem cell may be transformed into a cancer

stem cell through dysregulation of the proliferation and
differentiation pathways controlling it.
 Notch

 The Notch pathway has been known to

developmental biologists for decades.

 Its role in control of stem cell proliferation has now

been demonstrated for several cell types including
haematopoietic, neural and mammary stem cells.

 Components of the Notch pathway have been

proposed to act as oncogenes in mammary and other
tumors.
 The Notch signaling pathway is a highly conserved cell
signaling system present in most multicellular organisms.

 Notch is present in all metazoans, and vertebrates possess

four different notch receptors, referred to as Notch1,
Notch2, Notch3, and Notch4.

 The Notch receptor is a single transmembrane receptor

protein. It is a hetero-oligomer composed of a large
extracellular portion, which associates in a calcium-
dependent, non-covalent interaction with a smaller piece of
the Notch protein composed of a short extracellular region,
a single transmembrane, and a small intracellular region.
 Notch ligands are single-pass transmembrane
proteins.

 There are two classes of Notch ligands, the

Delta/Delta-like and the Serrate/Jagged class.
  The Notch receptor
protein sits like a trigger
spanning the cell
membrane, with part of it
inside and part outside.

 Ligand proteins binding

to the extracellular
domain induce proteolytic
cleavage and release of
the intracellular domain,
which enters the cell
nucleus to alter gene
expression.

 Because most ligands

are also transmembrane
proteins, the receptor is
normally triggered only
from direct cell-to-cell
Deltex-interacting protein contact.
 The Notch signaling pathway is important for cell-cell
communication, which involves gene regulation
mechanisms that control multiple cell differentiation
processes during embryonic and adult life.

 Notch signaling also has a role in the commitment to

the osteoblastic lineage, suggesting a potential
therapeutic role for Notch in bone regeneration and
osteoporosis.
Notch signaling pathway:
 In pancreatic cancer stem cells, notch signaling cascade plays a
vital role in stem cell maintenance and differentiation process.
 Notch receptor is composed of an extracellular ligand binding
domain, a single transmembrane spanning region and
intracellular domain.
Activation of notch signaling takes place through binding of delta
ligand with notch receptor between neighboring cells. Upon
ligand binding to notch receptor, it will undergo a conformational
change that allows cleavage at extracellular portion of notch by a
metalloprotease TNFα converting enzyme (TACE).
Subsequently, the intracellular portion of notch will also be
cleaved by delta-secretase, an intramembrane protease thereby
releasing notch intracellular domain containing portion (NICD).
In pancreatic cancer cells, NICD will translocate into the nucleus
and interacts with its transcription factor RBP and co activator
p300 leading to activation of Epcam, CD44 and Hes1 genes.
 knockdown of Hes1 using shRNA and inhibition of the
Notch pathway components by gamma secretase
resulted in the reduction of the self-renewal capacity of
pancreatic CSCs (Journal of Stem Cell Research, 2014)
 Wnt Signaling

 The Wnt signaling pathway describes a complex network

of proteins most well known for their roles in
embryogenesis and cancer, but also involved in normal
physiological processes in adult animals.

 Wnt proteins form a family of highly conserved secreted

signaling molecules.

 There are 19 human Wnt genes (Wnt1, -2, -2B,-3, -3A...).

Wnt target genes are for examples: c-myc, cyclin D, c-jun,

Runx2, nanog, Oct4.
 The name Wnt was coined as a combination of Wg
(wingless) and Int and can be pronounced as 'wint'.

 The wingless gene had originally been identified as a

recessive mutation affecting wing in Drosophila
melanogaster.

 The INT genes were originally identified as vertebrate

genes near several integration sites of mouse
mammary tumor virus (MMTV).

 The Int-1 gene and the wingless gene were found to be

homologous, with a common evolutionary origin
evidenced by similar amino acid sequences of their
encoded proteins.
 Mutations in Wnt genes or Wnt pathway components
lead to specific developmental defects, while various
human diseases, including cancer, are caused by
abnormal Wnt signaling.
 Wnt and Stem Cells

 Wnt proteins are important stem cell regulatory

factors.

 Wnts regulate maintenance and self renewal of

embryonic and adult stem cells.

 However, the roles of individual Wnts depend on the

identity of the Wnt gene and the specific stem cell
population.
 Traditionally, it is assumed that Wnt proteins can act
as Stem Cell Growth Factors, promoting the
maintenance and proliferation of stem cells.

 Wnt signaling was first identified as a potential

component to differentiation because of its established
role in development.

 There are data to suggest that Wnt signaling induces

differentiation of pluripotent stem cells into mesoderm
and endoderm progenitor cells.
 In adult stem cell differentiation, such as in
hematopoiesis, Wnt signaling controls the
maintenance and expansion of precursor populations.

 In contrast, in embryonic stem cells Wnt/β-catinin

signaling directs cells toward meso- and endodermal
lineages.
 Downstream regulators of the Wnt pathway such as
GSK (glycogen synthase kinase)-3β have proven
useful targets for the regulation of pluripotency in
cell culture using small molecule inhibitors.

 Additionally, the efficiency of iPS cell generation has

been improved with the in vitro addition of Wnt3a.

 TCF3, a transcription factor regulated by Wnt

signaling, has been shown to repress nanog, a gene
required for stem cell pluripotency and self-renewal.
 Wnt3 is associated with mesoderm and endoderm
differentiation.

 Wnt1 appears to be a major factor in self-renewal of

neural stem cells.
Wnt signaling can be divided into two categories:
Canonical and non-canonical Wnt signaling.

 Canonical Wnt signaling has been studied more

extensively than non-canonical Wnt signaling and
refers to a single clearly defined pathway, known as
the Wnt/β-catenin pathway.

 Non-canonical signaling encompasses multiple

pathways, the effects of which are mediated in a β-
catenin independent manner.
 Wnt Signaling

1. The Wnt pathway involves a large number of proteins that

can regulate the production of Wnt signaling molecules, their
interactions with receptors on target cells.

2. The presence and strength of any given effect depends on the

Wnt ligand, cell type, and organism.
3. Wnt proteins bind to cell-surface
receptors of the Frizzled family,
causing the receptors to activate
Dishevelled (DSH) family proteins.

4. Cell surface Frizzled (FRZ)

proteins usually interact with a
transmembrane protein called LRP.
LRP binds Frizzled, Wnt and axin
and may stabilize a
Wnt/Frizzled/LRP/Dishevelled/axin
complex at the cell surface.
5. In vertebrates, several
secreted proteins have been
described that can modulate
Wnt signaling by either
binding to Wnts or binding to
a Wnt receptor protein. For
example, Sclerostin can bind to
LRP and inhibits Wnt
signaling.

6. Dishevelled (DSH) is a key

component of a membrane-
associated Wnt receptor
complex which, when activated
by Wnt binding, inhibits a
second complex of proteins
that includes axin, GSK-3, and
the protein APC.
6. The axin/GSK-3/APC
complex normally promotes
the proteolytic degradation of
the β-catenin intracellular
signaling molecule.
7. The trimeric G proteins
can function downstream
from Frizzled. Wnt-
activated G proteins
participate in the
disassembly of the
axin/GSK3 complex.

8. Several protein kinases

and protein phosphatases
have been associated with
the ability of the cell
surface Wnt-activated Wnt
receptor complex to bind
axin and disassemble the
axin/GSK3 complex.
9. After this "β-catenin destruction
complex" is inhibited, a pool of
cytoplasmic β-catenin stabilizes,
and some β-catenin is able to enter
the nucleus and interact with
TCF/LEF family transcription
factors to promote specific gene
expression. (for maintenance of
self-renewal property).

OCT4
Nanog
Sox-2
 Phosphorylation of the
cytoplasmic domain of LRP
by CK1 can regulate axin
binding to LRP.

 Phosphorylation of β-catenin by GSK3

leads to the destruction of β-catenin.
How to activate or detect Wnt signaling in cells?

 Add Wnt protein, either in a purified form or as

conditioned medium to cells.

 most cells respond to Wnt signaling by an increase in

the levels of beta-catenin.
 There are good antibodies to detect beta-catenin by
Western Blots.
 There is also a monoclonal antibody to the non-
phosphorylated ("activated") form of beta-catenin.
 Mutations in Wnt genes or Wnt pathway components
lead to specific developmental defects, while various
human diseases, including cancer, are caused by
abnormal Wnt signaling.
 Wnt-1 was found as an
oncogene activated by
the Mouse Mammary
Tumor Virus in murine
breast cancer.

 Mutations in the
human AXIN1 gene
were reported in human
hepatocellular
carcinomas.
 TCF1 can also act as a
tumor suppressor gene,
as Tcf1 mutant mice
develop adenomas in the
gut and mammary
glands.