The Cancer Stem Cell Hypothesis

Traditionally, cancer has been viewed as a disease in which environmental or endogenous events induce mutations to critical oncogenes and tumor suppressor genes within a normal cell. The clinical manifestations of cancer appear when these mutations result in the cell’s tranformation to a more primitive, highly proliferative state from which the leukemia or solid tumor develops by clonal expansion.¹ This paradigm, however, does not readily explain the long developmental latency of many cancers and metastases, the mechanism by which the initiating events produce cellular dedifferentiation and cellular immortality, or the genesis of the functional and phenotypic diversity of the cells within the tumor itself. In the past decade, there is growing evidence that malignant tumors originate from the transformation of tissue stem cells by mutations that lead to the dysregulation of the normal mechanisms that control stem cell growth and proliferation.^1-9

Adult stem cells populate discrete niches within each organ. They are long-lived and multipotent in that they can recapitulate the entire range of cell types within that organ. Two salient qualities of stem cells are:
1) their ability to remain quiescent within the niche environment for long periods of time, and,
2) their capacity for asymmetric cell division giving rise to one stem cell, termed self-renewal, and one differentiated progeny.

In addition, stem cells are resistant to apoptosis, have enhanced telomerase and DNA repair activities, and have membrane-bound ABC transporters that exclude xenobiotics making them relatively resistant to the damaging effects of chemotherapeutic agents and other exogenous poisons.^9-11 The capacity for self-renewal coupled with long quiescent periods allows a stem cell exposed to damaging agents to accumulate mutations that may, over time, result in malignant transformation.³

In 1926, Bailey and Cushing proposed that cancer was initiated and maintained by a small number of transformed precursor cells.^10,12 However, it was only within the past decade that Dick and his coworkers first found strong evidence that all the cancer phenotypes present in acute myeloid leukemia (AML) were derived from a few rare (0.1-1% of the total cell population) leukemic stem cells.^2,4 These cells had the cell surface markers of normal hematopoietic stem cells, but had a much higher rate of self-renewal than normal stem cells and could identically reproduce the phenotypic diversity of human AML when injected into immune-compromised NOD/SCID mice.

More recently, small populations of stem-like cells have been isolated from most leukemias^2,4,13 as well as from many solid tumors such as brain glioblastomas and medulloblastomas,^4,5,10,14,15 and breast,^3,16 cervical, colorectal,^17,18 gastrointestinal, hepatocellular, lung, pancreatic, prostate, and skin carcinomas.^6,9,19,20 Cancer stem cells display the same cell surface markers as their normal counterparts (see Table 1), but demonstrate uncontrolled proliferation, perhaps due to a reduced responsiveness to negative growth regulators or to the loss of contact inhibition and gap junction intercellular communication (GJIC).^1,10

Table 1: Cancer Stem Cell Markers

A malignant tumor resembles a new organ composed of abnormally differentiated cells that show both genotypic and phenotypic diversity.^1,4,7 However, only the small populations of stem-like cells can form colonies in cell culture systems or engraft and recapitulate the entire diversity of the human tumor phenotype when injected into NOD/SCID mice. Furthermore, there is some evidence for functional diversity within the stem cell population of the tumor.

A subset of cancer stem cells, called side population, express the multidrug resistance transporters ABCB1 and ABCG2 and can be identified by their ability to exclude rhodamine and Hoechst dyes. There is evidence that these side population cells are more tumorigenic and have greater metastatic potential than other cancer stem-like cells; this may be due to an increased ability to survive traditional chemotherapeutic regimen.^15,18,19 In other studies a subset of precancerous stem cells that constitutively express VEGFR2 receptors have been identified within some tumor cell populations. These cells appear to be the source of the intrinsic vasculature and capillary beds of the tumor, and normal angiogenic processes may be involved only in attaching this intrinsic tumor vasculature to the body’s circulatory system.⁷

Unlike normal stem cells in which self-renewal terminates when the stem cell niche is replenished, the uncontrolled self-renewal of cancer stem cells and stem cell-derived cancer progenitor cells overpopulates the niche and infiltrates the surrounding tissue.¹³ Hedgehog, Notch, Wnt, and PTEN are some of the pathways that control the self-renewal, proliferation, and survival of both normal and cancer stem cells. Mutations leading to the constitutive activation of one or more of these pathways are observed in most aggressive cancers.

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Hedgehog and Bmi1

The Hedgehog (Hh) pathway (see Figure 1) regulates adult stem cell quiescence and self-renewal. Three Hh ligands have been identified in mammals, Sonic hedgehog (Shh), Desert hedgehog (Dhh), and Indian hedgehog (Ihh), of which Shh is the best studied. In the absence of ligand, the Hh receptor PTCH1 inhibits signaling through the catalytic inhibition of the transmembrane protein Smoothened (SMO). Ligand occupation of PTCH1 inactivates the receptor and allows activation of SMO that, in turn, results in the induction of Gli transcription factors; Gli1 and Gli2 are positive mediators of Hh signaling while Gli3 is a negative regulator.^3,20

Figure 1: Hedgehog pathway schematic. See text for further description.

Many of the effects of Hh activation are facilitated by the induction of Bmi1, a polycomb gene that represses transcription through chromatin remodeling and down-regulates the expression of genes in the Ink-4A/ADP ribosylation factor (ARF) complex, such as p16 Ink4A and p19 ARF, that are negative regulators of the cell cycle and are involved in stem cell quiescence and differentiation.^3,4 This enables stem cell proliferation and self-renewal via the Gli1- and Gli2-induced expression of the growth promoting genes cyclin D1, Myc, and Snail as well as upregulation of the Hh pathway elements PTCH1, Gli1, and Gli2.^19,20

Activation of the Hh pathway is seen in many carcinomas, including those from brain, stomach, pancreas, breast, prostate, lung, and skin.^9,19 Mutations that inactivate PTCH1 or that activate SMO are seen in basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma,^6,20 while aberrant Hh signaling is seen in some stomach, lung, and prostate carcinomas and in multiple myeloma.²⁰ There is a signature 11-gene expression profile associated with Hh-induced Bmi1 expression that correlates with poor prognosis in ten different types of human malignancies.^3,9 Inhibition of SMO can inhibit the growth and invasiveness of metastatic cancer cells in vitro and in vivo. The most widely studied SMO inhibitor, cyclopamine, has been shown to reduce the rate of proliferation of stem-like cancer cells and induce apoptotic death of metastatic cancer cells, but it has no effect on normal epithelial cells.^3,6

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The Notch-γ-Secretase Pathway

Notch signaling promotes the survival and proliferation of normal neural stem cells and inhibits their differentiation. Notch signaling is highly activated in stem-like cells from many cancer cell lines including T cell leukemias, as well as brain, breast, ovarian, cervical, colorectal, pancreatic, salivary gland, and lung carcinomas.^3,6,9,15 In particular, medulloblastoma and T cell lymphoblastic leukemia are Notch dependent malignancies.¹⁵

Notch activation (see Figure 2) involves the proteolytic cleavage of the Notch ligand/receptor complex by γ-secretase to release the Notch intracellular domain fragment (NICD) that translocates to the nucleus and upregulates expression of Myc, Hes1, and other genes.²² When the DAOY medulloblastoma cell line was transfected with NICD2 to make Notch signaling constitutively active, the transfected cells produced more xenograft tumors than the non-transformed DAOY cells and increased the population of both CD133⁺ and side population stem-like cells in culture. In contrast, inhibition of γ-secretase reduced the side population to 0.01% of the total cell count and inhibited by 90% the ability of cells to colonize soft agar or to form tumor xenografts in immune-compromised mice. NICD2 transfection protected the cells from the effects of γ-secretase inhibition.¹⁵ Thus, in some tumor types, the inhibition of Notch signaling can deplete a population of cells that are required for tumor initiation.

Figure 2: Notch pathway schematic. See text for further description.

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The Wnt-β-Catenin Pathway

The Wnt pathway promotes self-renewal, proliferation, and transient differentiation of normal stem cells and is involved in vertebrate limb development and regeneration.²³ It maintains the adult stem cell populations of tissues with rapid cellular turnover such as hair follicles, mammary gland, skin, and intestinal lining and often is constitutively activated in tumors arising in these adult stem cell niches.^17,23 This pathway also contributes to the malignant transformation of cancer stem and progenitor cells from stomach, pancreas, liver, prostate, brain, and lung, as well as some leukemias such as multiple myeloma and chromic myelogenous leukemia.^6,9,16,19

Wnt comprises a family of 19 extracellular glycoproteins that bind to the Frizzled (Fz) family of receptors, thereby activating a pathway that inhibits the proteolytic degradation of β-catenin. This allows the accumulation of cytoplasmic β-catenin and its translocation to the nucleus where it promotes gene transcription through the Tcf/Lef transcription factors. In the absence of Wnt activation, the proteins Axin and adenomatous polyposis coli protein (APC) form a complex with glycogen synthase kinase 3 (Gsk3) that promotes the phosphorylation of cytoplasmic β-catenin that, in turn, targets β-catenin for proteolytic degradation.

Activation of the Wnt pathway requires the association of Fz with its coreceptor, LDL receptor-like protein 5/6 (Lrp5/6). Formation of the Wnt/Fz/Lrp5/6 complex activates the phosphoprotein Dishevelled (Dvl), an integral component of the Fz receptor that, in turn, recruits Axin/Gsk3, thus dissociating the Axin/APC/Gsk3 complex. Lrp5/6 is sequentially phosphoryled by Gsk3 and casein kinase 1γ (Ck1γ). Phosphorylated Lrp5/6 binds to Axin/Gsk3, forming a complex that stabilizes β-catenin by preventing its phosphorylation (see Figure 3).

Figure 3: Wnt pathway schematic. See text for further description.

Mutations that either inactivate APC or damage the phosphorylation site of β-catenin will produce constitutive activation of β-catenin-induced gene transcription.^23,24 The majority of colorectal cancers involve either the loss of APC function or oncogenic mutations to β-catenin.¹⁷ Some of the genes upregulated by Wnt signaling include those that code for cyclin D1, Myc, survivin, osteopontin, tenascin C, and L1CAM. Inhibiting the transciption of Myc blocks the expression of approximately half the genes induced by Wnt signaling.^19,23

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The PTEN-mTOR Pathway

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor gene that maintains stem cells in a quiescent state.²⁵ It is a negative regulator of both STAT3 and mTor.18 mTor is a serine-threonine kinase that regulates pathways involved in protein synthesis and in cell growth and survival. STAT3 is a transcription factor that regulates genes involved in stem cell self-renewal.

When PTEN is mutated or deleted, there is an increased expression of genes related to cell cycle activation and DNA replication. These stem cells show enhanced self-renewal and maintain their multipotency, but proliferation is no longer responsive to negative growth factor control.²⁵ Deletion of PTEN is sufficient to convert a normal hematopoietic stem cell to the AML phenotype.⁵ Deletion of PTEN in a murine model of prostate cancer reproduces the disease progression seen in human prostate cancer.4 PTEN is also often mutated or deleted in glioblastoma.²⁵

Rapamycin inhibits mTor and is a downstream inhibitor of the PTEN pathway. If rapamycin is administered to mice soon after PTEN is deleted, the mice do not develop leukemia; if it is administered after the leukemia develops, the mice will live longer but will not be cured of the leukemia.^13,26

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Conclusions

Currently, cancer chemotherapy is deemed successful if it reduces tumor burden by blocking the proliferation and inducing apoptosis of cancer cells. However, often the cancer recurs or metastases develop at distant sites long after the primary tumor has been eradicated. Cancer stem cells are relatively rare. Like normal stem cells, they have the capacity to remain quiescent within a niche for long periods of time and become activated in a growth-permissive microenvironment. Like hematopoietic stem cells, they may be able to survive migration in the blood stream to distant niches. Much evidence has accumulated indicating that only this small population of cancer stem cells can engraft in immune-compromised mice, producing tumors that recapitulate the total phenotype of the original tumor.

Cancer stem cells appear to be controlled by pathways that are quiescent in normal adult cells, such as Hedgehog, Notch, Wnt, and PTEN. Targeting these pathways in addition to the pathways targeted by current chemotherapeutics may reduce disease recurrence and metastasis and may improve long-term survival rates.

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Materials

References

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