Stem Cells, Cell Differentiation, and Cancer

Properties of Normal Stem Cells

HSCs are the most studied and best understood type of adult stem cells and serve as a model for stem cells in other tissues. Hematopoiesis is a tightly regulated process in which a small pool of HSCs give rise to an increasingly diversified population of oligolineage intermediates, which in turn form the full repertoire of mature elements of the lymphohematopoietic system (e.g., erythrocytes, platelets, granulocytes, macrophages, and lymphocytes; see Fig. 7.1 ). These mature cell types perform a diverse set of functions, ensuring tissue oxygenation, blood coagulation, and immunity. HSCs have four fundamental properties: self-renewal, differentiation, migration, and homeostatic control. First, HSCs need to self-renew to maintain the stem cell pool. Self-renewal is not synonymous with proliferation. Self-renewal is a cell division in which one or both of the daughter cells remain undifferentiated and maintain the identity of a stem cell, endowed with unlimited growth potential. Second, HSCs must undergo differentiation: they must generate a progeny of cells that expand and progressively specialize in order to replenish the various populations of mature cells that are damaged, aged, or otherwise lost. Third, HSCs are able to enter the circulation and migrate from one blood-forming site to another (e.g., from the marrow of one bone to that of another) to replace HSCs that have been lost and maintain a constant output of blood cells. Finally, HSCs must balance self-renewal, differentiation, migration, and tissue repair according to a strict genetic program, which ensures homeostatic control of their numbers and which regulates their expansion in response to various types of stress, such as bleeding or infection.

In mice, HSCs represent less than 0.05% of bone marrow cells and are admixed with a heterogeneous pool of other immature progenitors. This pool includes a specific subset of immature multipotent progenitors (MPPs), which are able to differentiate into the full variety of hematopoietic lineages but lack self-renewal capacity. These populations form a hierarchy in which HSCs give rise to MPPs, which in turn give rise to oligolineage progenitors and the various mature cell types of the blood and lymphoid tissues (see Fig. 7.1 ). As HSCs mature to become MPPs, they increase their proliferation rate but lose the ability to self-renew. Only HSCs can reconstitute hematopoiesis in the long-term, for the lifetime of the animal, whereas MPPs can reconstitute hematopoiesis for a short period of time, usually less than 16 weeks.

A similar hierarchy is mirrored also in human blood tissues, wherein HSCs and MPPs are characterized by a CD34 ⁺ CD38 ⁻ phenotype and by the lack of mature blood cell lineage markers (Lin ⁻ ), but can be separated based on the differential expression of CD90 in HSCs (see Fig. 7.1 ). More generally, the hierarchic organization of hematopoietic tissues can be successfully applied to model the cellular composition and developmental dynamics of many adult mammalian tissues, such as the brain, mammary gland, skin, and intestinal epithelium.

Genetic Regulation of Self-Renewal

The long-term survival of a tissue, either normal or neoplastic, is dependent on its capacity to self-renew, whereas its overall size is determined by the balance between the rates of cell proliferation and cell death (or removal) across its various components. In normal tissues, stem cell numbers are under tight genetic regulation, resulting in the long-term maintenance of a constant tissue size. In contrast, tumor tissues have escaped this homeostatic regulation. Within a tumor, the number of cells with the ability to self-renew is constantly expanding, resulting in continuous tissue growth. It is not surprising therefore that many known oncogenes are able to expand stem cell numbers, either by protecting them from apoptosis or by inhibiting their differentiation. For example, enforced expression of Bcl2 results in an expansion of HSCs, whereas enforced expression of c -myb and c -myc prevents hematopoietic cell differentiation along the erythroid lineage.

Because the size of both normal and cancer tissues is dependent on the number of cells able to self-renew, it is also not surprising that a specific subset of oncogenes and/or tumor suppressor genes might directly activate and/or disable self-renewal pathways. Indeed, the basic molecular machinery that ensures the unlimited growth capacity of most cancer cells (e.g., the telomerase enzymatic complex) is fundamental to ensure self-renewal of most normal stem cells. The capacity to modulate self-renewal, however, is not a necessary attribute of all oncogenes and/or tumor suppressor genes: enforced expression of Bcl2, for instance, although able to expand the number of self-renewing HSCs, is unable per se to endow self-renewal properties on more mature cell types, such as MPPs.

Among cancer genes with direct control over self-renewal functions, the best example is probably the Bmi1 oncogene, which can cooperate with c- myc to induce B-cell lymphomas. Bmi1 is a member of the Polycomb repressor complex 1 (PRC1), a multiprotein transcriptional repression complex involved in the epigenetic regulation of developmental and differentiation processes. In the hematopoietic system, Bmi1 expression is highest in HSCs and gradually decreases as they differentiate into MPPs and oligolineage progenitors. In mice genetically deficient for Bmi1 ( Bmi1 ^{− / −} ) the number of HSCs is markedly reduced at birth, and their transplantation in lethally irradiated animals is able to reconstitute hematopoiesis, but only in a transient and self-limiting fashion, indicating a cell autonomous defect of self-renewal. Lack of Bmi1 expression causes similar phenotypes across many types of adult stem cells, including neural and mammary stem cells, and can be phenocopied by the overexpression of PRC1 inhibitors, such as the histone deubiquitinase Usp16. Important to note, lack of Bmi1 expression is also associated with upregulation of tumor suppressors, such as the cyclin-dependent kinase inhibitors encoded by the alternative transcripts of the Cdkn2a locus (i.e., the p16 ^Ink4a and p19 ^Arf proteins), and with overexpression of transcription factors that promote differentiation, such as selected members of the Hox family. This suggests that, in stem cells, the function of Bmi1 is to prevent the expression of a cascade of genes whose coordinated activity can lead to loss of self-renewal capacity. Remarkably, in a mouse model of leukemia initiated in Bmi1 ^{− / −} mice, leukemic cells are initially able to expand and cause the death of their primary hosts, but are unable to sustain long-term growth when transplanted into secondary syngeneic animals, where they eventually arrest, differentiate, and undergo apoptosis. Infection of Bmi1 ^{− / −} leukemic cells with a retrovirus encoding for Bmi1 completely rescues this defect, allowing leukemic cells to grow indefinitely and be serially passaged in mice. Similar observations have also been made in human breast cancer, where downregulation of BMI1 by means of enforced expression of microRNA-200c associates with reduced expansion and impaired tumorigenicity of cancer cells. Once more, this defect can be rescued by enforced expression of a modified version of the BMI1 mRNA, insensitive to the inhibitory action of microRNA-200c. Interesting to note, human BMI1 appears to induce telomerase activity. Again, in many human model systems, cancer cells lacking telomerase, although capable of substantial short-term proliferation and numeric expansion, progressively exhaust their growth capacity and irreversibly arrest. These studies reinforce the concept that in order to endow cancer cells with unlimited growth potential, a property frequently referred to as “immortality,” constitutive activation of proliferation pathways is not sufficient. It is also necessary to ensure the activation of self-renewal pathways, either directly (i.e., by activating genes that positively regulate them) or indirectly (i.e., by inactivating genes that negatively regulate them).

Many other signaling pathways implicated in oncogenesis are known to play central roles in stem cell biology, tissue morphogenesis and development. Classic examples among them are the Wnt, Notch, and Sonic hedgehog (SHH) pathways, all of which appear to regulate self-renewal functions in many normal tissues.

The Wnt pathway was first implicated in a mouse model of breast cancer, in which aberrant expression of Wnt1, caused by insertion of the mouse mammary tumor virus (MMTV) close to the Wnt1 gene, resulted in mammary tumors. Wnt1 belongs to a large family of secreted proteins that bind to receptors of both the Frizzled and low-density lipoprotein receptor–related protein (Lrp) families, resulting in activation of β-catenin. In the absence of Wnt stimulation, β-catenin is degraded by the adenomatous polyposis coli (Apc), glycogen synthase kinase-3β (Gsk3β), and Axin protein complex. Wnt/β-catenin signaling plays a pivotal role in the self-renewal of many normal stem cells. Activation of β-catenin signaling by Wnt proteins allows expansion of stem/progenitor cells, both in vitro and in vivo, and across different tissues, including the bone marrow, skin, mammary gland, and small intestinal epithelium. Constitutive activation of the β-catenin pathway is oncogenic and is almost invariably observed in colon cancer, most frequently by inactivating mutations of human APC . Current evidence suggests that β-catenin signaling is key for cancer cells to maintain a stem/progenitor cell phenotype. In colon cancer cells, inhibition of the β-catenin pathway induces expression of p21 ^cip1/waf1 , a cell-cycle inhibitor, followed by proliferation arrest and acquisition of a differentiated phenotype. Enforced expression of c- myc, an oncogene whose transcription is activated by β-catenin, inhibits p21 ^cip1/waf1 expression and allows cancer cells to continue proliferating in the absence of β-catenin signaling, revealing yet another role for a classic oncogene, c- myc, in the regulation of cell differentiation.

The Notch family of receptors, first identified as regulators of wing patterning in the Drosophila fruit fly, controls development and differentiation in many tissues, and across many animal species. In vitro stimulation of HSCs with selected Notch ligands (i.e., Jagged-1, Delta) transiently increases the activity of stem/progenitor cells, both in vitro and in vivo, suggesting that Notch activation promotes expansion of either HSCs or MPPs. Constitutive activation of the Notch pathway is endowed with powerful oncogenic effects in both mouse and human cells. The mouse oncogene int-3 encodes a constitutively active, truncated variant of the Notch-4 receptor. In humans, aberrant activation of NOTCH1, either by point mutation or chromosomal translocation, is a common occurrence in T-cell acute lymphoblastic leukemia (T-ALL). Important to note, inhibition of NOTCH-1 signaling can induce apoptosis in T-ALL cell lines, and is now being explored as a promising therapeutic strategy.

The SHH pathway provides yet another example of a pathway with key roles in tissue development, stem cell homeostasis, and oncogenesis. Like the Wnt factors, SHH is a secreted molecule and a powerful morphogen. SHH acts as a ligand for receptors encoded by members of the Patched gene family and activates signaling circuitries that regulate self-renewal in selected types of stem cells, such as bladder stem cells. Germline mutations in the SHH gene cause aberrations of embryo development in Drosophila and holoprosencephaly in humans. With regard to human cancer, germline mutations in Patched-1 ( PTCH1 ) cause Gorlin or basal cell nevus syndrome (BCNS), whereas sporadic mutations in PTCH1 are observed in the majority of sporadic basal cell carcinomas (BCCs) and a substantial fraction of medulloblastomas. Most importantly, pharmacologic inhibition of the SHH pathway has shown powerful antitumor activity in clinical trials, against both human BCCs and medulloblastomas harboring PTCH1 mutations, and is rapidly changing treatment guidelines for these diseases.

In conclusion, mouse genetic studies have contributed to the discovery of several gene families involved in either the positive or negative regulation of self-renewal properties in normal stem cell populations. Normal stem cells are characterized by the high expression of genes required to enable self renewal, and by the low expression of genes known to disable it. The observation that, in cancer cells, transforming mutations often target the same gene families, either by constitutively activating genes that promote self-renewal (Bmi1, Wnt, Notch, SHH ) or by inactivating genes that contribute to restrict it (Cdkn2a), suggests that stem cells and cancer cells depend on a common set of signaling pathways to control their numbers and stimulate their growth. The therapeutic success obtained with SHH inhibitors demonstrates how the study of stem cells and their self-renewal pathways can lead to novel and powerful antitumor treatments.