The phosphatidyl inositol 3-kinase (PI3K)/Akt pathway is activated downstream of a variety of extracellular signals and activation of this signaling pathway impacts a number of cellular processes including cell growth, proliferation and survival. The alteration of components of this pathway, through either activation of oncogenes or inactivation of tumor suppressors, disrupts a signaling equilibrium and can thus lead to cellular transformation. The frequent dysregulation of the PI3K/Akt pathway in human cancer has made components of this pathway attractive for therapeutic targeting; however, a more comprehensive understanding of the signaling intricacies is necessary to develop pharmacological agents to target not only specific molecules, but also specific functions. Here, we review a series of experiments examining the contribution of molecules of this signaling network including PI3K, phosphatase and tensin homolog deleted on chromosome 10, integrin-linked kinase and Akt and address the significance to human breast cancer.
The progression of primary mammary epithelial cells to a malignant phenotype involves multiple genetic events including the activation of oncogenes and inactivation of specific tumor suppressor genes. In this regard, activation of the phosphatidyl inositol 3-kinase (PI3K)/Akt pathway is thought to play a critical role in both the initiation and progression of human breast cancer. Activation of the PI3K pathway can occur in response to a variety of extracellular signals through engagement of either growth factor or integrin receptor pathways. Receptor-mediated activation of the PI3K pathway results through the recruitment of the p85 regulatory subunit of PI3K via its Src-homology 2 (SH2) domains to phophotyrosine residues located within the receptor. Once recruited to the membrane, the p110 catalytic subunit of PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) at the 3′ position of the inositol ring, thus generating PIP3. The resulting PIP3 serves to recruit phospholipid-binding domain containing proteins to the plasma membrane.
In particular, Akt and phosphoinositide-dependent kinase 1 (PDK1) are recruited to the membrane via their plekstrin homology (PH) domains, whereby PDK1 phosphorylates a site in the kinase domain of Akt (threonine 308 in Akt1). Phosphorylation at a second residue (serine 473 in Akt1) present within the regulatory carboxy-terminal region by a second kinase (PDK2) is required for full activation. The identity of PDK2 remains controversial and potential candidates include the integrin-linked kinase (ILK), mammalian target of rapamycin (mTOR)–rictor complex, protein kinase C (PKC)βII and Akt itself (Delcommenne et al., 1998; Lynch et al., 1999; Toker and Newton, 2000; Kawakami et al., 2004; Sarbassov et al., 2005). Upon activation, Akt moves to the cytoplasm and nucleus, where it phosphorylates a plethora of downstream targets. The activity of PI3K is opposed by the action of lipid phosphatases including phosphatase and tensin homolog deleted on chromosome 10 (PTEN), which removes the 3′ phosphate of PIP3, thus regenerating PIP2 and attenuating signaling downstream of activated PI3K. The coordinated regulation of PIP3 levels in the cell provide a means of intricately controlling downstream signaling activity that is essential to maintain the fine balance required for controlled cell growth.
More recently there is an increasing body of evidence to suggest that activation of the PI3K pathway can occur via activating mutations in the p110α catalytic subunit and may be an important contributing element to mammary tumor progression (Bachman et al., 2004; Samuels et al., 2004; Kang et al., 2005). These observations suggest that alteration of PI3K signaling may play a key role in the regulation of malignant breast cancer growth. In this review, we will highlight recent advances in understanding the role of this key signaling pathway in breast cancer progression.
The Akt/PKB family of serine kinases plays distinct roles in mammary tumor progression
The Akt family of serine–threonine kinases consists of three members, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ. The Akt family has been shown to be the primary downstream mediator of the effects of PI3K and regulates a variety of cellular processes through the phosphorylation of a wide spectrum of downstream substrates. Indeed, dysregulation of the PI3K/Akt signaling pathway can lead to an alteration of all the aspects of cell physiology that comprise the hallmarks of cancer (Hanahan and Weinberg, 2000) (Figure 1). Akt has been implicated in regulating the cell cycle through the phosphorylation and thus cytoplasmic retention of the cell cycle inhibitors p21 and p27 and through increased translation and stabilization of cyclin D1 protein levels (Diehl et al., 1998; Zhou et al., 2001; Viglietto et al., 2002). Akt also plays a role in metabolism by inactivating glycogen synthase kinase (GSK) and activating glycolysis and glucose transport (Cross et al., 1995; Kohn et al., 1996; Deprez et al., 1997). Cell survival is influenced by Akt through a variety of effector proteins including inhibition of the proapoptotic Bcl2 family member Bad and inhibition of the forkhead transcription factors which normally activate apoptotsis related genes (Datta et al., 1997; del Peso et al., 1997; Brunet et al., 1999). Furthermore, indirectly through the phosphorylation of IκB kinase (IKK) and directly through the activation of nuclear factor κB (NFκB) transcriptional activity, Akt leads to NFκB-mediated expression of prosurvival genes (Kane et al., 1999; Ozes et al., 1999; Romashkova and Makarov, 1999; Sizemore et al., 1999). Finally, Akt also plays a role in cell growth and translation through pathways leading to the activation of mTOR (Li et al., 2004).
Akt1 has been shown to contribute to tumor invasion and metastasis by promoting the secretion of matrix metalloproteinases and the induction of epithelial to mesenchymal transition (Thant et al., 2000; Larue and Bellacosa, 2005). Overexpression of Akt2 has been shown to upregulate β1-integrins and increase invasion of human breast and ovarian cancer cells (Arboleda et al., 2003). Recently, there has been increasing evidence that the Akt isoforms may regulate different cellular processes. Indeed, the knockout of individual isoforms in mice results in distinct phenotypes (Chen et al., 2001; Cho et al., 2001a, 2001b; Easton et al., 2005; Tschopp et al., 2005). One possible explanation for the differential behavior of these isoforms derives from the observation that in contrast to Akt1, Akt2 localizes predominantly adjacent to the collagen IV matrix during cellular attachment (Arboleda et al., 2003). The difference in localization may provide a mechanism for contrast in the processes regulated by the Akt isoforms. Overexpression of only the Akt2 isoform has been shown to duplicate the invasive effects of PI3K-transfected breast cancer cells and the expression of a kinase dead Akt2, but not Akt1 or 3, prevents invasion induced by either activation of PI3K or overexpression of Neu (Arboleda et al., 2003). Together, these data indicate that Akt2, among the members of the Akt family, may have particular importance in mediating PI3K-dependent effects on cellular adhesion, motility, invasion and metastasis.
Subsequent studies suggest that the different Akt family members play opposing roles in terms of breast cancer cell motility and invasion. Indeed, two separate studies have implicated Akt1 as an inhibitor of cell motility and invasion. The Brugge laboratory used an MCF10A breast epithelial cell line expressing the insulin-like growth factor-insulin receptor (IGF-IR) in transwell motility assays to assess the contribution of Akt1 and Akt2 to cell migration (Irie et al., 2005). In this model they demonstrated that the downregulation of Akt1 using short-hairpin RNA dramatically increased cell migration. In contrast, the downregulation of Akt2 did not affect migration and in fact, the concomitant downregulation of Akt2 and Akt1 abrogated the migratory effect of Akt1 knockdown, suggesting that Akt2 is required for this phenotype. They further demonstrated that the downregulation of Akt1 increased ERK activation and conclude that Akt1-mediated suppression of ERK signaling is responsible for the antimigratory effect of Akt1.
A second group has also demonstrated an anti-migratory function for Akt1, although through a mechanism independent of ERK signaling (Yoeli-Lerner et al., 2005). Overexpression of Akt1 in breast cancer cell lines resulted in a decrease in both migration and invasion, whereas siRNA knockdown produced an increase in migration and invasion. They further demonstrated that this effect was mediated through the proteasomal degradation of the nuclear factor of activated T cells (NFAT) transcription factor, via Akt-mediated activation of the Mdm2 ubiquitin ligase. It therefore appears that Akt1-dependent inhibition of migration can be achieved by at least two distinct mechanisms.
Consistent with these tissue culture experiments, studies with transgenic mice have also provided evidence to support the concept that Akt isoforms may play distinct roles in breast cancer progression. Mammary-specific expression of an activated form of Akt1 possessing aspartic acid substitutions at key phosphorylation sites (Akt1-DD) was not by itself sufficient to induce mammary tumors (Hutchinson et al., 2001). However, transgenic mice expressing this activated form of Akt1 exhibited a profound defect in mammary gland involution owing to suppression of the normal apoptotic program in the regressing mammary epithelium (Hutchinson et al., 2001). Consistent with these observations, mammary-specific expression of a myristylated form of Akt1 or wild-type Akt1 resulted in a profound delay in mammary involution (Schwertfeger et al., 2001, 2003; Ackler et al., 2002). Mammary epithelial expression of a constitutively activated Akt1 could dramatically accelerate mammary tumor growth in mice expressing a mutant polyoma virus mT oncogene (PyV mT) uncoupled from the PI3K pathway (Hutchinson et al., 2001). Despite this dramatic rescue of tumor growth in these mutant PyV mT strains, mammary-specific expression of activated Akt1 could not rescue the potent metastatic phenotype associated with expression of the wildtype PyV mT oncogene (Hutchinson et al., 2001). Similarly, the coexpression of activated Akt1 with an activated ErbB2 receptor in the mouse mammary epithelium accelerated tumor onset (Hutchinson et al., 2004). Interestingly, expression of activated Akt1 completely abrogated the ability of the activated ErbB2 tumor cells to form secondary lesions in the lungs of tumor bearing mice. This is consistent with, and compliments the results observed in the previously mentioned cell culture systems whereby Akt1 plays an anti-invasive role. Taken together, these observations suggest that activation of Akt-1, although not sufficient to induce mammary tumors, can cooperate with other oncogenic events to accelerate tumor induction. Future studies with transgenic mouse models bearing either gain-of-function or loss-of-function alleles of Akt family members should allow these issues to be addressed.
Whereas these experimental observations suggest that the Akt family play an important role in mammary tumorigenesis, activating mutations of the Akt family in human cancer have not been reported. Akt2 amplification has been described in some tumor types with one study reporting genomic amplification in 3% of breast carcinomas, as well as 12% of ovarian tumors (Bellacosa et al., 1995). This study also found that Akt2 amplification was more frequent in undifferentiated ovarian tumors (4/8), suggesting an association with tumor aggressiveness. Even though the Akt1 gene is rarely amplified, Akt1 protein levels and activity have been reported to be elevated in some types of cancer. Immunohistochemical staining of a series of breast cancers in one study revealed elevated Akt1 staining in 24% of tumors, whereas strong Akt2 staining was evident in only 4% of tumors (Stal et al., 2003). Another study reported increased Akt1 kinase activity in about 40% of breast cancers (Sun et al., 2001). However, the expression of Akt2, but not Akt1, has been correlated with Her2 expression in breast cancer tissues, and the ectopic expression of Her2 in a breast cancer cell line resulted in upregulation of Akt2 (Bellacosa et al., 1995). Although Akt3 has been less extensively studied, there is evidence that it may also be involved in tumorigenesis. It has been shown that Akt3 overexpression inversely correlates with estrogen receptor status in human breast cancers (Nakatani et al., 1999). Furthermore, an extra arm of chromosome 1q is a recurrent genomic alteration observed in a variety of human cancers and given the location of the akt3 gene in this region, it is likely that Akt3 copy number is increased in a subset of tumors. Of concern in terms of therapeutics, Akt activation has been shown to contribute to radiation and chemotherapy resistance. In this regard, it is interesting to note that the combination of small molecule inhibitors of the PI3K/Akt pathway with standard chemotherapy in experimental studies has proven successful in diminishing chemotherapeutic resistance (West et al., 2002).
The opposing roles played by different Akt family members highlight the importance of using isoform specific antibodies when evaluating cellular processes attributed to the Akt family as such may not hold true for all three family members. Similarly, caution should be exercised when evaluating human tumor samples by immunohistochemical staining, paying careful attention to the contribution of individual isoforms. In terms of developing novel therapeutics, the inhibition of Akt1 may provide desirable outcome in terms of arresting tumor cell proliferation; however, in light of recent work, this may also inadvertently lead to the dissemination of tumor cells and exacerbate the disease state. The accumulating evidence implying a distinct role for the different Akt isoforms highlights the importance of determining the substrates unique to each isoform, which will allow for the therapeutic targeting of specific aspects of tumorigenesis more effectively.
Mutational alteration of PI3K and PTEN in mammary tumor progression
Alteration of the PI3K pathway is common in cancer and aberrations in components of this pathway have been demonstrated in a variety of different human cancers (reviewed in Hennessy et al. (2005). The PI3K itself is a target of activating mutations (Bachman et al., 2004; Samuels et al., 2004; Kang et al., 2005) and indeed the most common genetic aberration in breast cancer is somatic mutation of the PIK3CA gene encoding the p110α catalytic subunit. In a large study of human breast tumor samples and cell lines performed to evaluate PIK3CA mutations, 77 of 292 (26%) primary breast tumors and 14 of 50 (28%) breast cancer cell lines displayed mutations in the PIK3CA gene (Saal et al., 2005). These results were consistent with a previous study in which a mutation rate of 22% in a set of 41 breast tumors and 30% in 12 cell lines was reported (Bachman et al., 2004). In both of these studies, the mutations were found to cluster in two previously reported ‘hotspot’ regions in exons 9 and 20, corresponding to the helical and catalytic domains of p110α, respectively. The first study found three mutations in exon 9 and eight in exon 20 in 53 breast tumor samples (Bachman et al., 2004), whereas 31 and 49 mutations were detected in exons 9 and 20, respectively by the second group (Saal et al., 2005). Unlike the predominance of exon 9 mutations in colorectal cancer (Samuels et al., 2004), the results of these studies suggest that exon 20 mutations in the PIK3CA gene are most common in breast cancer.
In addition to the activation of the catalytic subunit of PI3K, there is compelling evidence implicating the mutational inactivation of PTEN phosphatase in human breast cancer. Indeed, PTEN inactivation was first noted in inherited breast cancer disease known as Cowden's syndrome (Dahia et al., 1997; Liaw et al., 1997; Nelen et al., 1997; Rhei et al., 1997; Steck et al., 1997). In addition to its documented role in inherited forms of human breast cancer, there have been reports of PTEN mutations in sporadic breast cancer (Teng et al., 1997; Garcia et al., 1999; Minobe et al., 1999). Immunohistochemical analysis of PTEN status in breast tumors has revealed a highly significant association with PIK3CA mutations, whereby PIK3CA mutations were found for the most part in PTEN+ tissues. Expanding on these observations, sequencing of the PTEN gene further improved the inverse correlation between PIK3CA mutations and disrupted PTEN when including both PTEN− and mutated PTEN. The infrequent overlap between PIK3CA mutations and abrogated PTEN is not surprising, given that the two proteins catalyse opposing reactions. This suggests that once PIK3CA is activated or PTEN lost or mutated, there is a diminished selective advantage for targeting the other gene given that both mutations result in an increase in PIP3.
Direct evidence implicating loss of PTEN activity in causal induction of mammary tumors derives from both germline and conditional knockouts of PTEN in a number of animal model systems. For example, germline knockout mice heterozygous for PTEN loss develop mammary tumors resembling those induced in Cowden's syndrome (Stambolic et al., 2000). In addition, germline loss of one PTEN allele has also been shown to accelerate Wnt1-induced mammary carcinogenesis (Li et al., 2001). Finally, mammary-specific ablation of a conditional PTEN allele has been demonstrated to result in the induction of mammary neoplasia (Li et al., 2002). Although the molecular mechanism of mammary tumor induction in these models remains to be elucidated, there are recent data in prostate systems to suggest that one possible mechanism for acceleration of tumor growth is an expansion of the cancer stem cell compartment (Wang et al., 2006). Whether a similar cancer stem cell expansion occurs in these breast cancer models awaits further experimental examination.
Integrin-mediated activation of PI3K pathway
The ILK was originally identified through its association with the intracellular domains of β1 and β3 integrin subunits (Hannigan et al., 1996). ILK is a 59-kDa protein consisting of N-terminal ankyrin repeats, a central plekstrin homology domain and an atypical serine/threonine kinase domain in the C-terminus (Hannigan et al., 1996). By associating with multiple cytoplasmic scaffolding proteins, such as paxillin and actopaxin, ILK provides an important link between integrins and the actin-based cytoskeleton (Tu et al., 2001; Wu, 2001; Yamaji et al., 2001; Brakebusch and Fassler, 2003). In addition, ILK is linked indirectly to growth factor receptors, such as the platelet-derived growth factor receptor, through adaptor molecules including PINCH and Nck2 (Tu et al., 2001; Wu, 2001). Genetic and biochemical studies have revealed important roles for ILK in regulating several aspects of epithelial cell biology, including proliferation, survival and adhesion (Hannigan et al., 1996; Wu et al., 1998; Attwell et al., 2000; D'Amico et al., 2000; Persad et al., 2000). These properties of ILK have been attributed both to its role as a scaffolding protein, as well as to the serine/threonine kinase activity localized to the C-terminus.
One of the principal downstream targets of ILK kinase activity was shown to be Akt (Delcommenne et al., 1998). Following overexpression of ILK in cultured epithelial cells, Akt was found to be robustly phosphorylated on ser473 (Delcommenne et al., 1998). In addition, plating of cells on a physiological substrate, such as fibronectin, or addition of growth factors such as insulin, resulted in Akt phosphorylation which could be abrogated by introduction of a kinase-dead ILK allele (Hannigan et al., 1996; Delcommenne et al., 1998). Biochemical analysis involving in vitro kinase assays subsequently demonstrated that ILK could phosphorylate Akt directly on ser473, suggesting that ILK was functioning as PDK2 (Delcommenne et al., 1998; Persad et al., 2001).
The experimental evidence implicating ILK in Akt regulation was strengthened by the observation that ILK kinase activity was induced by phosphoinositide lipid products of PI3K, and could be blocked by overexpression of PTEN (Delcommenne et al., 1998; Persad et al., 2000). Conversely, mutations in PTEN resulted in constitutive activation of both ILK kinase activity and Akt phosphorylation (Persad et al., 2000). Importantly, it was shown that the PI3K-induced phosphorylation of Akt could be blocked by expression of kinase-dead ILK (Delcommenne et al., 1998).
Although the results of these experiments suggested that ILK was important for PI3K-mediated phosphorylation of Akt, the biological relevance of these observations remained uncertain (Zervas and Brown, 2002). Much of this concern was based on observations in lower organisms, where the ablation of ILK had no effect on Akt phosphorylation (Zervas et al., 2001; Mackinnon et al., 2002). Nonetheless, there is ample evidence from human cancer cell lines and mouse tumor models that ILK plays a critical role in the PI3K/Akt1 pathway during oncogenic transformation and tumor progression. A role for ILK in tumor progression is suggested by the observation that ILK expression correlates with high tumor grade and poor prognosis in a variety of human tumors (Chung et al., 1998; Graff et al., 2001; Marotta et al., 2001; Ahmed et al., 2003; Dai et al., 2003). In some cases, such as in human colon cancer, the upregulation of ILK protein levels corresponds to an increase in Akt phosphorylation (Bravou et al., 2006).
More direct evidence for ILK in the tumor-promoting properties of PI3K/Akt signaling, however, comes from the manipulation of human cancer cell lines and mouse models. For example, the inhibition of ILK kinase activity in PTEN-mutant prostate and glioblastoma cells results in apoptosis and reduced tumor growth in vivo (Persad et al., 2000; Edwards et al., 2005; Koul et al., 2005). The impact of ILK inhibition in these cells was due to downregulation of Akt phosphorylation, and is consistent with reports that ILK suppresses anoikis through activation of Akt (Attwell et al., 2000). Similarly, we have found that the targeted ablation of ILK in Neu-induced mammary tumor models results in a dramatic reduction in Akt phosphorylation and impaired tumor growth (unpublished observations). Conversely, the transgenic overexpression of ILK in the mouse mammary gland epithelium results in a hyperplastic phenotype and subsequent tumor formation, which is associated with increased levels of Akt phosphorylation (White et al., 2001).
In addition, ILK has been shown to induce vascular endothelial growth factor expression in human prostate cancer cells through Akt1-mTOR-mediated stabilization of hypoxia inducible factor-1 (HIF-1) (Tan et al., 2004). As a result, blocking ILK kinase activity in a prostate xenograft tumor model results in delayed tumor growth owing to inhibition of angiogenesis (Tan et al., 2004). Similar results have also been reported for a xenograft model of thyroid cancer, where ILK has been found to be upregulated during the transition to an anaplastic phenotype (Younes et al., 2005). In this case, blocking ILK kinase activity was also associated with a decrease in Akt phosphorylation, accompanied by a reduction in cell survival, angiogenesis and tumor growth (Younes et al., 2005). These models therefore strongly implicate a critical role for the PI3K/ILK/Akt pathway in the growth of human tumors.
The studies outlined above illustrate the significant progress that has been made understanding the importance of the PI3K signaling network in breast cancer progression. The use of transgenic mouse models, where the different components of PI3K pathway have been altered, have been instrumental in elucidating the molecular basis for the potent transforming activity of the PI3K network. Although no single genetically engineered mouse can offer a complete model of the wide assortment of neoplasms found in human breast cancer, it is hoped that these multiple approaches will enable us to develop insights into the complex molecular events involved in tumorigenic progression of the breast. More recently, the combination of these techniques to create bigenics, as well as the use of ‘knock-in’ and conditional tissue-specific gene targeting strategies, have allowed the creation of models more reflective of the human disease to be devised. Ultimately, these mouse models will serve as important tools to assess the efficacy of various therapeutic agents directed against various component of the PI3K signaling network.
WJM is supported by CRC Chair in Molecular Oncology. RLD is supported by a pre-doctoral award from the DOD. This work was supported by grants from NCI PPG #102036 and CBCRA awarded to WJM.
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Role of PI3K/Akt on migration and invasion of MCF10A cells treated with extracellular vesicles from MDA-MB-231 cells stimulated with linoleic acid
Journal of Cell Communication and Signaling (2019)