STEM CELLS IN SOLID TUMORS CANCER STEM CELL (CSC)

Cancer stem cell theory The idea of cancer cells arising from a common origin has been thoroughly explained and published as the Unitarian or Trophoblastic theory of cancer in The idea of cancer cells arising from a common origin has been thoroughly explained and published as the Unitarian or Trophoblastic theory of cancer in 1950.Unitarian or Trophoblastic theory of cancerUnitarian or Trophoblastic theory of cancer It states that cancer--differentiated trophoblast proliferation-- is part of the healing process, and the disease only manifests if its control (immune response and nutrition) are impeded. It states that cancer--differentiated trophoblast proliferation-- is part of the healing process, and the disease only manifests if its control (immune response and nutrition) are impeded.immune response nutritionimmune response nutrition

Cancer stem cell theory There are two competing visions of tumors. Old cancer model: 1) All tumor cells can form new tumors and are therefore equally tumorigenic. 1) All tumor cells can form new tumors and are therefore equally tumorigenic. 2) Unregulated growth is due to serial acquisition of genetic events leading to the expression of genes that promote cell proliferation with concomitant silencing of growth inhibitory genes and blunting of cell death. 2) Unregulated growth is due to serial acquisition of genetic events leading to the expression of genes that promote cell proliferation with concomitant silencing of growth inhibitory genes and blunting of cell death. 3) Cancer is a proliferative disease. 3) Cancer is a proliferative disease.

Cancer stem cell theory New cancer model: 1) Tumors arise from cells termed cancer stem cells that have properties of normal stem cells, particularly self- renewal and multipotentiality (a minority) of tumor cells. 1) Tumors arise from cells termed cancer stem cells that have properties of normal stem cells, particularly self- renewal and multipotentiality (a minority) of tumor cells. 2) Unregulated cell growth is due to a disruption in the regulatory mechanism in stem cell renewal. 3) Cancer is a stem cell disorder and not a simple mechanism whereby cell proliferation is disrupted. 3) Cancer is a stem cell disorder and not a simple mechanism whereby cell proliferation is disrupted.

Cancer stem cell theory These CSCs cells persist in tumors as a distinct population that likely causes relapses and metastasis. These CSCs cells persist in tumors as a distinct population that likely causes relapses and metastasis. This theory explains why are many cancers so difficult to treat. This theory explains why are many cancers so difficult to treat.

Cancer stem cell theory Why stem cells? Only stem cells have the ability to self renew and neoplasia is essentially dysregulated self renewal Only stem cells have the ability to self renew and neoplasia is essentially dysregulated self renewal Stem cells are long-lived cells which can acquire the necessary number of sequential mutations to convert a normal cell into a malignant one. Stem cells are long-lived cells which can acquire the necessary number of sequential mutations to convert a normal cell into a malignant one.

Are we targeting the right cells? Conventional chemotherapies kill differentiated or differentiating cells, which form the bulk of the tumor but are unable to generate a new one. Conventional chemotherapies kill differentiated or differentiating cells, which form the bulk of the tumor but are unable to generate a new one.chemotherapies A population of CSCs, which gave rise to it, remains untouched and may cause a relapse of the disease. A population of CSCs, which gave rise to it, remains untouched and may cause a relapse of the disease. Development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease, where little progress has been made in recent years. Development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease, where little progress has been made in recent years.metastatic diseasemetastatic disease

WRONG TARGET. Traditional cancer therapies (top) kill rapidly dividing tumor cells (blue) but may spare stem cells (yellow) that can give rise to a new tumor. In theory, killing cancer stem cells (bottom) should halt a tumor's growth lead to its disappearance.

What are Cancer Stem Cells? Cells that have properties of normal stem cells: 1) The abilities to self-renew. 1) The abilities to self-renew. 2) Tha ability to differentiate into multiple cell types. 2) Tha ability to differentiate into multiple cell types. 3) They form a distinct population in tumors that likely causes disease relapse and metastasis. 3) They form a distinct population in tumors that likely causes disease relapse and metastasis.relapse metastasisrelapse metastasis

Self-renewal of stem cells The concept of self- renewal is crucial to understand CSC, and also to get insight on the mechanism by which current therapies might evade the available treatments. The concept of self- renewal is crucial to understand CSC, and also to get insight on the mechanism by which current therapies might evade the available treatments.

Self-renewal of stem cells 1) Provides the cell with the ability to undergo infinite cellular divisions with only few of the stem cells dividing at a particular time. 2) The doubling time of most stem cells is relatively long, as compared to their immediate progenitors, which replicate with shorter doubling times (Repair of DNA damage). 3) In some stem cells at division the `mother’ cell retain the original chromosome while providing the daughter with the newly formed chromosome (Chromosomal preservation) → minimizes mutation in the mother cell.

CSC Development The molecular pathways for stem cell differentiation are complex indicating that dysregulation could occur at multiple sites to turn off the homeostatic balance and create abnormal cells, or cancer cells, also referred as malignant cells or transformed cells. The molecular pathways for stem cell differentiation are complex indicating that dysregulation could occur at multiple sites to turn off the homeostatic balance and create abnormal cells, or cancer cells, also referred as malignant cells or transformed cells.

Normal Stem Cells vs. Cancer Stem Cells The stem cells in tumors (CSCs) are not the same type of stem cells being explored as potential therapies to treat degenerative diseases. The stem cells in tumors (CSCs) are not the same type of stem cells being explored as potential therapies to treat degenerative diseases. But they develop because of mutations that accumulate over years and often decades. The mutations are thought to promote the tumor stem cells' ability to proliferate, eventually leading to cancer But they develop because of mutations that accumulate over years and often decades. The mutations are thought to promote the tumor stem cells' ability to proliferate, eventually leading to cancer

Properties shared by normal stem cells and cancer stem cells Assymetric Division: Assymetric Division: –Self renewal Tissue-specific normal stem cells must self-renew throughout the lifetime of the animal to maintain specific organs Tissue-specific normal stem cells must self-renew throughout the lifetime of the animal to maintain specific organs Cancer stem cells undergo self-renewal to maintain tumor growth Cancer stem cells undergo self-renewal to maintain tumor growth –Differentiation into phenotypically diverse mature cell types Give rise to a heterogeneous population of cells that compose the organ or the tumor but lack the ability for unlimited proliferation (hierarchical arrangement of cells) Give rise to a heterogeneous population of cells that compose the organ or the tumor but lack the ability for unlimited proliferation (hierarchical arrangement of cells) Regulated by similar pathways Regulated by similar pathways –Pathways that regulate self-renewal in normal stem cells are dys-regulated in cancer stem cells

Evidence for the presence of CSC 1) In exp. Animal research, efficient tumor formation to establish a tumor. This was formerly explained by: ** Poor methodology (loos of cell viability during transfer). ** Poor methodology (loos of cell viability during transfer). ** The critical importance of the microenvironment. ** The particular biochemical surroundings of the injected cells. ** The critical importance of the microenvironment. ** The particular biochemical surroundings of the injected cells. According to CSC theory only a small fraction of the injected cells, the CSC, have the potential to generate a tumor. In human AML the frequency of these cells is less than 1 in 10,000. only a small fraction of the injected cells, the CSC, have the potential to generate a tumor. In human AML the frequency of these cells is less than 1 in 10,000.

Evidence for cancer stem cells 2) Tumor heterogeneity: Most tumors are very heterogeneous and heterogeneity is commonly retained by tumor metastases. This implies that the cell that produced them had the capacity to generate multiple cell types (have a multidifferentiative potential), a classical hallmark of stem cells. This implies that the cell that produced them had the capacity to generate multiple cell types (have a multidifferentiative potential), a classical hallmark of stem cells.

CSC- PATHWAYS A normal stem cell may be transformed into a cancer stem cell through disregulation of the proliferation and differentiation pathways controlling it. A normal stem cell may be transformed into a cancer stem cell through disregulation of the proliferation and differentiation pathways controlling it.stem cellpathwaysstem cellpathways The first findings in this area were made using haematopoietic stem cells (HSCs) and their transformed counterparts in leukemia. The first findings in this area were made using haematopoietic stem cells (HSCs) and their transformed counterparts in leukemia. haematopoietic stem cellsleukemia haematopoietic stem cellsleukemia However, these pathways appear to be shared by stem cells of all organs. However, these pathways appear to be shared by stem cells of all organs.

CSC- PATHWAYS Bmi-1 This group of transcriptional repressor was discovered as a common oncogene activated in lymphoma and later shown to specifically regulate HSCs and neural stem cells. This pathway appears to be active in CSC of pediatric brain tumors and CRC This group of transcriptional repressor was discovered as a common oncogene activated in lymphoma and later shown to specifically regulate HSCs and neural stem cells. This pathway appears to be active in CSC of pediatric brain tumors and CRCtranscriptionalrepressoroncogenelymphomapediatricbrain tumorstranscriptionalrepressoroncogenelymphomapediatricbrain tumors

CSC- PATHWAYS Bmi-1 ** In normal cells BMI-1 inhibits the transcription of CDNK2A which encodes two cyclin dependent kinase inhibitors, INK4A and ARF. ** In normal cells BMI-1 inhibits the transcription of CDNK2A which encodes two cyclin dependent kinase inhibitors, INK4A and ARF. ** Cell cycle progression is promoted in the absence of INK4A and pro-apoptotic genes are inhibited in the absence of ARF. Hence, BMI-1 regulates proliferation and apoptosis. **In the case of cancer, BMI-1 is circumvented and CDNK2A is no longer inhibited, thereby resulting in unregulated proliferation and self-renewal.

CSC- PATHWAYS Notch The Notch pathway has been known to developmental biologists for decades. The Notch pathway has been known to developmental biologists for decades.Notch pathwayNotch pathway Its role in control of stem cell proliferation has now been demonstrated for several cell types including haematopoietic, neural and mammary stem cells. 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. Components of the Notch pathway have been proposed to act as oncogenes in mammary and other tumors.

CSC- PATHWAYS Wnt/β-catenine This pathway is strongly implicated as stem cell regulators. This pathway is strongly implicated as stem cell regulators. It is commonly hyperactivated in tumors and is required to sustain tumor growth. It is commonly hyperactivated in tumors and is required to sustain tumor growth. Their role has been illustrated especially in gliomas (the Gli transcription factors), CRC and mammary tumors. Their role has been illustrated especially in gliomas (the Gli transcription factors), CRC and mammary tumors.

The Wnt-B-catenine pathway In the normal Wnt pathway the levels of the transcription factor ß-catenin mediates self- renewal. In the normal Wnt pathway the levels of the transcription factor ß-catenin mediates self- renewal. ß-catenin could be turned off by a destruction complex as a feedback mechanism. ß-catenin could be turned off by a destruction complex as a feedback mechanism. However, in cancer the control process is circumvented and ß-catenin levels constantly thrive, hence causing continual proliferation and self- renewal. However, in cancer the control process is circumvented and ß-catenin levels constantly thrive, hence causing continual proliferation and self- renewal.

Wnt, Shh, and Notch pathways have been shown to contribute to the self-renewal of stem cells and/or progenitors in a variety of organs, including the haematopoietic and nervous systems. When dysregulated, these pathways can contribute to oncogenesis. Mutations of these pathways have been associated with a number of human tumours, including colon carcinoma and epidermal tumours (Wnt), medulloblastoma and basal cell carcinoma (Shh), and T-cell leukaemias (Notch). Pathways involved in self-renewal that are deregulated in cancer cells

Two General Models for Cancer Heterogeneity 1. All cancer cells are potential cancer stem cells but have a low probability of proliferation in clonogenic assays 2. Only a small definable subset of cancer cells are cancer stem cells that have the ability to proliferate indefinitely.

Which cells in the hierarchy are cancer stem cells?

Passegue, Emmanuelle et al. (2003) Proc. Natl. Acad. Sci. USA 100, Hematopoietic and Progenitor Cell Lines

Self-renewal Assay in NOD/SCID Mice (Non-obese diabetic/severe combined immunodeficiency) Sublethally irradiated NOD/SCID Mice FACS Cell Sorter Cancer Cells (ex: Leukaemia cells

Human hematopoietic cells are organized in a hierarchy that is sustained by a small population of self-renewing hematopoietic stem cells (HSCs). HSCs give rise to progressively more lineage- restricted, differentiated progenitors with reduced self-renewal capacity (LTC-ICs, long-term culture-initiating cells; CFU, colony-forming units), which in turn produce functionally mature blood cells. Disruption of pathways regulating self-renewal and differentiation through the acquisition of transforming mutations generates leukemic stem cells (LSCs) capable of sustaining growth of the leukemic clone in vivo. LSCs possess an altered differentiation program, as demonstrated by aberrant expression of some cell-surface markers (indicated in blue) and give rise to an aberrant developmental hierarchy that retains aspects of its normal counterpart. In vivo reconstitution assays using immune-deficient mouse recipients enable detection of HSCs and LSCs as SCID-repopulating cells (SRCs) and SCID leukemia-initiating cells (SL-ICs), respectively. Hierarchies in normal and leukemic human hematopoietic cells Wang and Dick 2005 Trends in Cell Biology 15:

Acute myeloid leukemia (AML) – CD34 + CD38 - Leukaemic Mouse Models: chronic myelomonocytic leukaemia (CMML)MRP8-BCL-2 acute myeloid leukaemia (AML)MRP8-BCL2Xlpr/lpr chronic myeloid leukaemia (CML)/BlastMRP8-PML-RAR α acute promyelocytic leukaemia (APML)77MRP8-BCRablXBCL-2 Hematopoietic Cancer Stem Cells

Self-renewal is a key property of both normal and leukemic stem cells. Fewer mutagenic changes are required to transform stem cells in which the self-renewal machinery is already active (a), as compared with committed progenitors in which self-renewal must be activated ectopically (b). In addition, self-renewing stem cells are long-lived; thus, there is an increased chance for genetic changes to accumulate in individual stem cells in comparison with more mature, short-lived progenitors. If a committed progenitor with limited life span acquires a genetic mutation that does not confer increased self-renewal (c), that cell will likely die or undergo terminal differentiation before enough mutations occur to propagate a full leukemogenic program. The importance of self-renewal in leukemic initiation and progression.

Brain Tumor Stem Cells: CD133 + CD133 – neuronal stem cell marker Brain tumor stem cells were identified from human brain tumor samples by in vitro neurosphere assays normally used to isolate normal neural stem cells Singh et. al 2003 Cancer Research 63: GFAP = glial fibrillary acidic protein

Brain tumor stem cells were identified by intracranial transplantation of CD133 + cells into adult NOD/SCID mouse forebrain. CD133 + CD133 - Singh et al Nature 432:

Breast Cancer Stem Cells: CD44 + CD24 low Lin - B ESA + CD44 and CD24 – adhesion molecules B38.1 – breast/ovarian cancer-specific marker ESA – epithelial specific antigen Al-Hajj, Muhammad et al. (2003) Proc. Natl. Acad. Sci. USA 100,

CSCs in different solid tumors Stem cells may cause some forms of bone cancer, University of Florida Osteosarcoma occurs right next to the most active centers of growth, the growth plates in long bones. Osteosarcoma occurs right next to the most active centers of growth, the growth plates in long bones. These areas of the skeleton contain many stem cells undergoing rapid growth and developing into bone during the adolescent growth spurt. These areas of the skeleton contain many stem cells undergoing rapid growth and developing into bone during the adolescent growth spurt. A stimulated, abnormal stem cell might therefore be the cell of origin of osteosarcoma. A stimulated, abnormal stem cell might therefore be the cell of origin of osteosarcoma. stem-like cells were isolated from tumors. About one in 1,000 cells in the samples had features of embryonic stem cells. The researchers also found abundant levels of the two key factors that help maintain embryonic stem cells in a very primitive state. stem-like cells were isolated from tumors. About one in 1,000 cells in the samples had features of embryonic stem cells. The researchers also found abundant levels of the two key factors that help maintain embryonic stem cells in a very primitive state.

The Real Problem in Breast Tumors: Cancer Stem Cells At the University of Michigan researchers have identified a small population of cells in breast tumors that can seed the growth of new cancers. These cancer- causing cells, which make up a tiny fraction of cells within tumors, have properties similar to those of stem cells. At the University of Michigan researchers have identified a small population of cells in breast tumors that can seed the growth of new cancers. These cancer- causing cells, which make up a tiny fraction of cells within tumors, have properties similar to those of stem cells.

CSCs in Colorectal carcinoma

CSCs in Hepatocellular carcinoma

CSC hypothesis & Drug Resistance The CSC hypothesis states: “the cancer-initiating cell is a transformed tissue stem cell, which retains the essential property of self-protection through the activity of multiple drug resistance transporters. The CSC hypothesis states: “the cancer-initiating cell is a transformed tissue stem cell, which retains the essential property of self-protection through the activity of multiple drug resistance transporters. This resting constitutively drug-resistant cell remains at low frequency among a heterogeneous tumor mass. This resting constitutively drug-resistant cell remains at low frequency among a heterogeneous tumor mass. The mutation allows for unbridled cell growth and resistance to chemotherapeutic efforts since CSCs express genes for drug resistance and anti-apoptotic mechanism,. The mutation allows for unbridled cell growth and resistance to chemotherapeutic efforts since CSCs express genes for drug resistance and anti-apoptotic mechanism,.

CONCLUSION Stem cells are immature cells that can replicate, or renew themselves, and are able to differentiate into all cells types. Stem cells are immature cells that can replicate, or renew themselves, and are able to differentiate into all cells types. mutations and rearrangements of the genomes of stem cells give rise to CSCs. These changes could underlie the development of cancers in many tissues. mutations and rearrangements of the genomes of stem cells give rise to CSCs. These changes could underlie the development of cancers in many tissues. Stem cells are more difficult to kill. Because they are so important throughout a person's lifetime, they have developed mechanisms that protect themselves. Therefore, tumor stem cells are able to resist toxic substances, such as cancer drugs. Stem cells are more difficult to kill. Because they are so important throughout a person's lifetime, they have developed mechanisms that protect themselves. Therefore, tumor stem cells are able to resist toxic substances, such as cancer drugs.

CONCLUSION The next step is to figure out what makes the CSC different from the other cells in the tumor. The next step is to figure out what makes the CSC different from the other cells in the tumor. DNA microarrays could be used to identify genes that are active in the cancer-causing cells (CSCs) compared to other tumor cells. Some of these genes might control the cell's ability to replicate and metastasize. DNA microarrays could be used to identify genes that are active in the cancer-causing cells (CSCs) compared to other tumor cells. Some of these genes might control the cell's ability to replicate and metastasize. Identifying these genes may suggest new drug targets that could selectively kill the cancer cells Identifying these genes may suggest new drug targets that could selectively kill the cancer cells