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Asymmetric stem cell division has emerged as a major regulatory mechanism for physiologic control of stem cell numbers. Reinvigoration of the cancer stem cell theory suggests that tumorigenesis may be regulated by maintaining the balance between asymmetric and symmetric cell division. Therefore, mutations affecting this balance could result in aberrant expansion of stem cells. Although a number of molecules have been implicated in regulation of asymmetric stem cell division, here, we highlight known tumor suppressors with established roles in this process. While a subset of these tumor suppressors were originally defined in developmental contexts, recent investigations reveal they are also lost or mutated in human cancers. Mutations in tumor suppressors involved in asymmetric stem cell division provide mechanisms by which cancer stem cells can hyperproliferate and offer an intriguing new focus for understanding cancer biology. Our discussion of this emerging research area derives insight from a frontier area of basic science and links these discoveries to human tumorigenesis. This highlights an important new focus for understanding the mechanism underlying expansion of cancer stem cells in driving tumorigenesis.
The ability of stem cells to undergo asymmetric cell division as a way to self-renew is a tightly regulated process that occurs during development, tissue maintenance, regeneration, and may be disrupted in hyperproliferative disease states such as cancer. Asymmetric cell division is typically restricted to stem cell populations where a need exists to preserve both a progenitor and a simultaneously generated differentiated progeny. This process is nicely exemplified in self-renewing tissues such as the epithelial layer of the human skin and intestine. Both organs possess stem cell pools that derive differentiated epithelia needed to maintain the function of the organ. The stem cell employs asymmetric division to maintain an appropriate census of daughter cells (or transient amplifying cells) and terminally differentiated cells. In the intestine, transient amplifying cells are characterized by their ability to amplify the epithelial population and likely undergo symmetric cell division to generate terminally differentiated cell populations. In these self-renewing tissues, a critical balance between asymmetric and symmetric cell division is required to maintain tissue homeostasis. Asymmetric stem cell division is vital for this maintenance thus it is likely that tumors will develop if it is not properly regulated (Figure 1A) . This dysregulation is consistent with the notion that expansion of a subpopulation of cancer cells harboring stem cell-like properties (cancer stem cells) may be the basis for propagating tumorigenesis. Though still controversial, there is growing acceptance that tumors may be dictated by this stem cell hierarchy, knowledge of how mutations in molecules that influence asymmetric stem cell division will provide insight into tumorigenesis. Mechanisms of asymmetric stem cell division have primarily been elucidated in invertebrate systems and encompass a number of molecules highly conserved in vertebrates [1-4].
A subset of proteins important for regulating asymmetric stem cell division are known tumor suppressors expressed in both invertebrate and vertebrate systems. Many of these tumor suppressors in Drosophila melanogaster have a role in tumor formation, and many of these genes have human homologues. Whether the function of these tumor suppressors in asymmetric stem cell division significantly contributes to cancer progression in vertebrate systems is not yet fully established, however based upon evolutionary conservation, it is intriguing to speculate that they may play an important role in human cancers.
Our intention is to review the role of four specific tumor suppressors involved in asymmetric stem cell division and discuss potential roles for their function in human cancer. Importantly, the lessons learned from the consequences of misregulating asymmetric stem cell division in developmental systems can inform the emerging research focus on the mechanism underlying cancer stem cell expansion as it relates to tumor progression.
Much of what is known about the molecular mechanism underlying asymmetric cell division is based on examination of developmental systems in Drosophila. Through elegant studies of the Drosophila neuroblast and germline cells, we know that the polarity and spindle orientation of the stem cell—the major determinants of asymmetric stem cell division—are governed by factors that are both intrinsic and extrinsic [2-5]. For example, in the fly neural stem cells or “neuroblasts,” segregation of intracellular proteins to the apical or basolateral region of the cell determines whether a stem cell asymmetrically divides, giving rise to both a stem cell and a differentiated daughter cell, or if it symmetrically divides to produce two stem cells [6, 7]. In contrast, Drosophila germline cells rely on extrinsic factors within the stem cell niche to define the orientation of the mitotic spindle, which is critical for proper cell division [8, 9]. In addition, proper cell division of the self-renewing, polarized mouse intestinal epithelium is regulated but the positioning of the mitotic spindle . From these two model systems, it is clear that both the orientation of the spindle and segregation of polarity components ultimately determine whether a stem cell will asymmetrically or symmetrically divide.
While it is yet unclear if these two well-defined examples of asymmetric stem cell division directly apply to division of human stem cells or to a putative cancer stem cell population, they offer a testable model to shape our investigation of the link between mammalian cancer and stem cell biology. Moreover, emerging evidence from the fly and mouse suggests that mutations in particular tumor suppressors that govern asymmetric stem cell division disrupt the normal ratio of stem cells to differentiated cells and contribute to unregulated proliferation of tumors. The following tumor suppressors - Adenomatous polyposis coli (Apc), Lethal giant larvae (Lgl), Brain tumor (Brat) and p63 - display a novel and intriguing link between invertebrates and vertebrates and the regulation of asymmetric stem cell division in both tissue stem cells and tumorigenesis.
APC is a human tumor suppressor with a well-documented role in the Wnt signaling pathway, primarily governing proliferative division of stem cells [11, 12]. In this capacity mutations in APC result in upregulated cellular proliferation and tumor formation. Not surprisingly, this gene is mutated or suppressed in a number of cancers, including hepatoblastoma , medulloblastoma , adult T-cell leukemia  and most notably in colorectal cancer where nearly all forms harbor mutations in APC . The dominant association between APC and the Wnt signaling pathway overshadows the multiple functions of the APC protein (reviewed in ). The human APC gene encodes a large multi-domain protein that can interact with a number of partner proteins . As such, APC has been demonstrated to interact with microtubules, suggesting a role in cell migration [17-19]. Additionally, it has been shown to be involved in regulating mitotic spindle assembly and chromosome segregation . Further, APC has also been described to participate in regulation of cell cycle progression and apoptosis [18, 21]. Although each of these roles attributed to APC have potential importance in tumor progression, its role in asymmetric stem cell division most intriguingly suggests an important function in modulating expansion of cancer stem cells. Recently, Apc was discovered to be a component of the centrosome complex of Drosophila germline cells. Here it functions in establishing asymmetric stem cell division, distinct from its role in Wnt signaling. In this capacity, mutations in APC could effectively expand early cancer cells or a putative cancer stem cell pool.
In the male Drosophila germline, Apc2 anchors the mother centrosome of the germline stem cell so that it is adjacent to the hub cell. The hub cell provides extrinsic, supportive cues to the stem cell that orient the mitotic spindle. The contact surface between the hub cell and the germline stem cell is also marked by concentrated levels of proteins known to interact with Apc2 (Figure 1B). This interaction localizes Apc2 to the interface between the hub germline stem cell, fixing the mother centrosome at one pole of the stem cell and allowing the daughter centrosome to migrate to the opposite side of the germline stem cell (Figure 1B). This movement allows the correct orientation of the mitotic spindle [9, 22]. In Drosophila, deletion of both Apc genes results in misorientation of centrosomes and the mitotic spindle, and consequently disrupts asymmetric stem cell division. At the tissue level, the phenotypic consequence is hyperproliferation of germline stem cells at the expense of differentiated cells .
To date, the most well investigated role of APC has been its function as a tumor suppressor. However, these new findings in Drosophila reveal a novel role for Apc in asymmetric stem cell division, which may intensify its role in tumor progression. It is easy to speculate that APC in humans may also be essential for spindle orientation and proper asymmetric division of tissue stem cells. In this scenario, mutations in APC would also lead to increased numbers of cancer stem cells. Investigation of APC's role in the regulation of asymmetric stem cell division represents an important future focus of human cancer biology.
Normal asymmetric neuroblast division results in self-renewal of the stem cell and production of a differentiated ganglion mother cell (GMC; Figure 1C). The correct positioning of both apical and basal protein complexes is critical for proper asymmetric cell division. Mutations in tumor suppressors cause mislocalization of these basal proteins, generating two neuroblasts that result in uncontrolled stem cell proliferation (Figure 1C). Three Drosophila tumor suppressor genes, lethal giant larvae (lgl), discs-large (dlg) and scribble (scrib), act in a common pathway in the mitotic neuroblast to establish the asymmetrically localized cortical basal complex and ultimately influence asymmetric stem cell division. Specifically, Lgl interacts with the apical complex shown in Figure 1C – atypical protein kinase C-Bazooka-Par6, Inscuteable, Partner of Inscuteable, and Gαi (aPKC, Ins, Pins and Gαi). In addition, it restricts the localization of active basal complex molecules [23, 24] and this is illustrated in mutants for Lgl that possess abnormal targeting of the basal complex proteins Miranda, Prospero and Numb (Mira, Pros, Numb) (Figure 1C; [6, 24]).
Proper location and function of Lgl is critical to the prevention of tumor formation. Lgl mutants do not asymmetrically divide but instead produce two stem cells at the expense of neuronal populations. This ultimately leads to expansion of the stem cell population and subsequent formation of brain tumors . Supporting this role in tumor suppression and the regulation of proper asymmetric cell division, loss of mouse Lgl1 results in disrupted asymmetric cell division and a brain tumor phenotype . Tumor initiation and cancer progression in humans may, in part, be driven by misregulation of Lgl homologues. Strikingly, a human homologue of lgl, HUGL-1, is lost in many solid tumors and is strongly correlated with advanced stages of malignant melanoma, colorectal cancer and endometrial cancer [26-28]. In addition, recent work examining HUGL-1 in human hepatocellular carcinoma reveals that the mRNA is frequently mutated by aberrant splicing. This renders the protein inactive and functions as an important mediator of hepatocellular carcinoma progression .
HUGL-1 is highly structurally and functionally conserved, as observed by the ability of HUGL-1 to rescue Drosophila lgl mutants . Both HUGL-1 and another Lgl human homologue, HUGL-2, directly interact with the human aPKC-Par6 protein complex  (Figure 1C), behaving just as Lgl does in Drosophila. Further, inhibition of this binding induces disorganization of the mitotic spindle during normal mitosis and results in aberrant cell division . Even further evidence from the examination of the behavior of Drosophila polarity proteins in human ovarian cancer epithelium reveals that Lgl function is conserved  between flies and humans. This suggests that both HUGL-1 and HUGL-2 play active roles in establishing polarity and spindle orientation for asymmetric cell division of human epithelial stem cells and, when mutated, may result in tumor initiation and/or expansion of cancer stem cells. This intriguing possibility has yet to be directly investigated.
The Drosophila larval neuroblast tumor suppressor with emerging relevance to human cancer is the newest member of the basal complex, “Brain tumor” (brat, Figure 1C). Brat, like Prospero, Miranda, and Numb, is asymmetrically localized during neuroblast cell division to the basal cortex (Figure 1C). Brat removal results in extensive proliferation of larval neuroblasts at the expense of differentiated neurons and generates tumors [33, 34]. Further investigation revealed that Brat mutants exhibit and uncontrolled expansion of transient amplifying cells, resulting from a failure to progress through the cell cycle .
The structure of Brat contains clues about its role in asymmetric stem cell division and control of tissue proliferation. Brat contains an NHL domain, an evolutionarily conserved motif found in proteins that post-transcriptionally regulate gene expression via mRNA binding and inhibition of translation. Indeed, in Drosophila, Brat is thought to be a post-transcriptional inhibitor of Myc . Myc transcriptionally regulates a variety of cell cycle and cell growth genes to support proliferation as opposed to differentiation. Interestingly, dMyc translation is de-repressed in cells without Brat. Studies in the mouse neocortex support this finding where TRIM32, a Brat homologue, is asymmetrically localized in one of the two daughter cells and becomes upregulated during neuronal differentiation . TRIM32 has dual roles as both a tumor suppressor functioning to degrade cMyc, and in asymmetric cell division where it is asymmetrically located and activates certain microRNAs important for stem cell self-renewal .
Although a role for TRIM32 in asymmetric stem cell division in human cancers has not been described, its overexpression in human head and neck squamous cell carcinoma samples highlights its activity as an E3 ubiquitin ligase [38-41], and it has been suggested to potentially be a cause of cancer. This analysis is complicated by functional redundancy in the mouse and human systems, as both species also express TRIM2 and TRIM3, which are also human Brat homologues. To this end, loss of heterozygosity of the tumor suppressor TRIM3 has recently been implicated human malignant gliomas , where it is intriguing to speculate that it may have a role in asymmetric cell division and formation of a potential cancer stem cell population. Clearly, additional studies are required to establish a role for the Brat family homologues in asymmetric stem cell division in humans. However, the intriguing evidence from mouse and fly suggests that Brat is certainly multi-functional. Importantly, like the APC tumor suppressor, Brat is most well-known for an early-described function in tumorigenesis that overshadows a potentially important role in asymmetric stem cell division specifically contributing to cancer stem cell expansion.
While numerous studies have examined asymmetric cell division in invertebrates, recent reports have uncovered a role for this process in mammalian systems. For example, p63, a closely related member of the p53 tumor suppressor family, is highly expressed in asymmetrically dividing stratified epithelial cells that are susceptible to cancer . p63 has been implicated and is now classically described as a master switch regulator of epithelial stem cell commitment, maintenance, and differentiation .
In both mouse and human, full length p63 is spliced into multiple isoforms and all of the resulting protein products have distinct, complex roles described in both oncogenesis and tumor suppression depending on the tissue and/or tumor-specific context for which they are examined [44, 45]. For example, ΔNp63α is upregulated in breast, gastric, cholangiocarcinoma, and chronic myeloid leukemia, but not in leukemia, where the TAp63 isoform is overexpressed. In addition, molecular interactions between the isoforms, which limit the activity of one another have also been described further complicating analyses of these proteins .
Interestingly, the p63 transcription factor was recently implicated in mitotic spindle orientation during asymmetric epidermal stem cell division, one of the first studies to examine in vivo asymmetric stem cell division in a mammalian system . Accordingly, its role in establishing spindle orientation may be related to the proliferative potential of dividing cells, a critical mediator in cancer progression. Stratified epithelial cells undergo symmetric and asymmetric divisions during normal development. Under these conditions, the mitotic spindle orients perpendicular to the basement membrane and mammalian homologues of the apical complex localize to the apical cortex prior to cell division. In contrast, in a p63 null mouse mutant, the spindle is parallel in orientation and the complex is mislocalized , resulting in the disruption of tissue organization.
In general, misregulation of any of the p63 isoforms is implicated in human tumor formation, however a definitive and consistent correlation between expression of the p63 isoforms and either cancer initiation or progression remains controversial [44, 45]. Many human cancers harbor an overexpression of one or more isoforms of p63, supporting an oncogenic role in tumorigenesis. In addition, some studies indicate that overexpression of p63 can cause an epithelial-to-mesenchymal transition, leading to an upregulation of genes involved in cell migration and invasion during metastasis . Interestingly and conversely, human bladder and urothelial cancers exhibit loss of p63, supporting a tumor suppressive role [48, 49]. In either situation and in light of the mouse and fly data, it is possible that either over- or underexpression of p63 may contribute to aberrant spindle orientation in the stem cell population. Clearly, the existence of multiple p63 isoforms complicates our appreciation of its role in asymmetric stem cell division, although the extensive descriptions of the function of p63 in epithelial stem cell biology combined with the recent spindle orientation studies in the mouse  provides an undeniable basis for further examination of the role of p63 in spindle orientation as it results in asymmetric stem cell division.
In any self-renewing tissue, maintaining a balance between stem cells and their differentiated progeny depends upon tightly regulated asymmetric stem cell division . Recent investigations in invertebrate and vertebrate systems have established that successful asymmetric cell division is dependent on asymmetrically localized proteins and mitotic spindle orientation. Proper execution of these two cellular programs functions in cell fate determination to control whether a cell assumes either a stem or differentiated identity. When mutations occur in genes involved either directly or downstream of the intrinsic or extrinsic cues governing these mechanisms, the resulting asymmetric cell division is abnormal and leads to uncontrolled amplification of stem cell populations.
The tumor suppressor proteins described in this review represent just four examples of molecules that regulate developmental or adult homeostatic asymmetric cell division, but we highlight the critical need for further investigations regarding the relevance of these tumor suppressors in disrupting asymmetric stem cell division in human cancer. Indeed, a precedent for such translational research is ongoing in work addressing Drosophila cell polarity determinants and proliferation control, and their implications on mammalian cancer progression [5, 30, 32, 50-52]. Studies emerging from this nascent field may implicate a critical and novel role for these proteins in early tumor initiation and asymmetric stem cell division of a putative cancer stem cell population.
Direct links between tumorigenesis and asymmetric stem cell division do exist in mutants of the well-documented mammalian tumor suppressor APC, the p53 family member, p63, and the Drosophila polarity protein, Lgl (summarized in Table 1). Mutations in these tumor suppressors are associated with advanced tumor progression, metastasis and poor patient prognosis, yet their function in aberrant asymmetric stem cell division in cancer has not been fully explored despite the amount of overwhelming evidence derived from lessons in developmental biology. The findings we present here likely represent but a few examples of how clues from developmental biology can illuminate key insights into cancer cell biology; specifically, the study of aberrant asymmetric stem cell division of human tissue stem cells and the expansion of a cancer stem cell pool in tumorigenesis. Clearly defining a role for the underlying mechanism driving aberrant expansion of a cancer stem cell pool provides further justification for targeting the cancer stem cell as an important therapeutic approach for treatment of disease.
We would like to thank Dr. Rachel Dresbeck and Dr. Chris Doe for critical review of the manuscript. This work was supported by grants to M.H.W. from the NIH DK068326, CA118235 and A.E.P. T32HD409309.
Competing Financial Interests: The authors declare no competing financial interests.
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