Dichotomy effects of Akt signaling in breast cancer

The serine/threonine kinase Akt/PKB has emerged as one of the most pivotal protein kinase family that plays critical roles in regulating pleiotropic cellular and physiological processes [1]. In response to ligand stimulation from cytokines or from growth factors such as the insulin-like growth factor-I (IGF-I) and the epidermal growth factor (EGF) family, receptor tyrosine kinases are phosphorylated, an event which subsequently activates phosphatidylinositol 3-kinase (PI3K) signaling and stimulates the Akt axis as well as other downstream signaling pathways [1]. To commence this signaling cascade, the lipid second messenger phosphatidylinositol (3,4,5)-triphosphate (PIP3) is first synthesized from PIP2 by PI3K and then recruits both Akt and phophoinositide-dependent kinase 1 (PDK1) to the plasma membrane through the pleckstrin homology (PH) domain where the highly conserved Thr308 is phosphorylated by PDK1 [2, 3]. Phosphorylation of this residue as well as Ser473, catalyzed by the mammalian target of rapamycin complex 2 (mTORC2), together confers full activation of Akt [4], thereby activating various downstream factors by phosphorylating arrays of targets [1]. Conversely, this activation cascade can be blocked by cellular inhibitors including the phosphatase and tensin homolog (PTEN) and INPP4B which directly antagonize PI3K function via dephosphorylating PIP3, thereby abrogating PIP3-mediated activation of downstream signaling events such as PDK1 and Akt [5, 6]. As a result, target cells can be returned to a basal level in a standby mode. However, in vitro engineered Akt kinase can override this regulatory mechanism and maintain it in a “supercharged” stage. This can be done by insertion of myristoylated (Myr) tag at its N-terminus which results in anchoring Akt in plasma membrane anchorage as well as constitutive activating Akt independently of PI3K activity [7, 8].

Three main Akt isoforms, Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ, have been discovered in mammals and they share two highly conserved regions in the PH and kinase catalytic domain [1]. Studies employing gene ablation in laboratory animals have revealed non-redundant functions of Akt isoforms. Akt1 null mice are small with significant growth defects [9, 10], whereas mice depleted of Akt2 develop insulin-resistant diabetes [11]. Conversely, Akt3 ablation leads to reduced brain size in mice [12, 13]. Notably, differences in physiology and microenvironment might exist between mice and humans. Clinical studies demonstrated that specific isoforms can be amplified in different types of human cancer, furthering a notion that Akt kinase dictates transformation phenotypes of various carcinomas in an isoform-specific manner, rather than in a redundant fashion [14]. Yet, how etiological cause(s) determine which of the 3 isoforms shall be activated and subsequently transmit unique downstream targets to exert distinct outcomes remains largely unknown.

Furthermore, components within the PI3K pathway are frequently dysregulated in human cancers [15]. For instance, activating mutations of PIK3C (the catalytic subunit of PI3K) often occur in prevalent carcinomas [16]. Moreover, PTEN has been proven to be one of the most commonly altered genes in human malignancies [17]. In contrast, gain-of-function Akt mutations are relatively uncommon [15, 17] and most frequently occur at residue 17 (namely E17K) which resides in the PH domain and is thus unlikely to directly sustain kinase activation. Based on clinical studies, it is becoming doubtful that Akt activation per se is indeed important for driving various neoplastic phenotypes. In support of this notion, activated Akt signaling was previously shown to induce senescence as well as inhibit breast cancer cell motility and invasion [18‐21].

Among its known neoplastic features, Akt kinase is involved in EMT, which is characterized by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype [22]. In carcinomas, EMT is associated with increased aggressiveness, tumor invasion, and metastatic potential, and endows mammary stem cell properties [23‐25]. A recent study demonstrated that Akt activation via down-regulated PTEN can enrich normal as well as malignant human mammary stem/progenitor cells and these aberrations can be rescued by Akt inhibitors [26]. Nevertheless, a mounting body of evidence supports the idea that Akt signaling regulates cell migration and EMT via an isoform-specific and context-dependent manner [27‐30]. It remains largely unclear whether Akt kinase would result in different outcomes, in respect to normal versus malignant breast epithelia. Moreover, it remains puzzling as to whether Akt activation augments a whole array of transformation phenotypes collectively leading to oncogenesis, or if it exerts paradoxical effects on both promoting and impeding neoplastic phenotypes.

To investigate these issues, we have expressed all three isoforms of constitutively active Myr-Akt kinase in human mammary epithelia ranging from nonmalignant primary epithelia, an immortalized cell line, and a series of cell lines exhibiting varying degrees of malignant behavior. This wide array of target cells has allowed us to reveal how Akt influences oncogenic phenotypic changes corresponding to the cell context in varying degrees of malignancy. We have discovered that Akt, in an isoform-independent fashion, has tumor suppressive properties since it can inhibit of EMT, decrease cell motility, and reduce the stem/progenitor cell population. These aberrations are rather prominent in non-malignant epithelia but diminish as cells progress to a more neoplastic state. However, even in non-malignant cells, Akt activation can have tumor-promoting properties since it can promote cell survival following exposure to chemotherapeutic agents. Taken together, this study denotes a novel paradigm that activated Akt signaling can have both tumor-suppressing and tumor-promoting properties.

A growing body of evidence has demonstrated that activation of components within the PI3K cascade are associated with human carcinomas including colon, endometrium, prostate, brain, ovarian, and breast cancers [15, 48]. However, gain-of-function mutations leading to constitutive activation of Akt are relatively uncommon (E17K is the most frequent occurrence) [15, 17], which contradicts a general belief that Akt activation plays critical roles in driving neoplastic phenotypes. In breast cancer, the oncogenic roles associated with Akt isoforms are still unclear and the discrepancies may be due to factors associated with mouse models vs. clinical studies, knockdown vs. overexpression, and in vitro vs. in vivo[27‐30, 32, 49]. Akt1 has been shown to inhibit EMT as well as cell motility and these aberrations can be rescued by Akt2, so that the net balance and ratio between the two isoforms dictated the overall cell fate [27, 29]. However, this observation raised two unresolved issues: (a) what upstream mediators, if any, would select which isoforms to be activated; and (b) what downstream targets can be uniquely transmitted in response to different isoforms and exert distinct and perhaps opposing effects. In the current report, we demonstrate that Akt isoforms appear to have redundant, rather than unique functions, when promoting neoplastic features.

We have identified Akt1 as being the major isoform in all breast epithelial cells examined in the current report (Additional file 1: Figure S4). In other independent studies, however, activation of Akt1 was demonstrated to suppress EMT [27], an event also important for stem cell self-renewal [23, 29]. Taken together, these findings suggest that upregulated HER2 or knocked down PTEN would have not only activated Akt1 signaling but also repressed EMT and subsequently lowered stem/progenitor subfraction. However, this rationale is contradictory to data reported in previous publications [26, 42]. Nevertheless, our present findings suggest an unreported paradigm that all Akt isoforms are likely to behave similarly for repressing cell migration, EMT, and stem/progenitor function, rather than exerting antagonistic effects by interacting among various isoforms. Two possibilities may explain the discrepancies between our data and others. Not only does the malignant state and cell context dictate Akt’s inhibitory effects (Figure 6), but the published findings are mainly generated from Akt knockdown studies that might induce unexpected feedback signaling. Notably, loss of Akt may not be physiologically relevant since most human carcinomas are associated with activation, rather than an inhibition, of Akt signaling. To date, Akt ablation has not been reported for any of the known human malignancies. Regarding isoform specificity, our current data cannot completely exclude the possibility that other undisclosed downstream targets or events might respond to Akt activation in an isoform-specific manner. However, this issue is beyond the scope of our current study.

The inhibition of cell migration and EMT by Myr-Akt in the current study appears to recapitulate some of the aberrations induced by PIK3CA mutations. This notion is supported by a clinical study from a large cohort of breast tumors (N = 590) in which the women who carried activation mutations of PIK3C also displayed improved prognosis, prolonged breast cancer-specific and overall survival as well as lymph node negativity [32]. However, our finding that Myr-Akt overexpression failed to expand stem/progenitor cell subpopulation is somewhat inconsistent with the data from exogenous expression of HER2 or from knockdown of PTEN by ShRNA [26, 42]. We reason the discrepancy is likely due to the fact that dysregulated HER2 and PTEN can trigger far broader downstream targets beside Akt. For instance, other than acting as a phosphatase to attenuate activated Akt, PTEN can regulate cell cycle progression, stem cell self-renewal, chromosome stability, and senescence [50]. Likewise, additional signaling cascades downstream from PI3K include mitogen-activated protein kinase (MAPK), extracellular signal-related kinase (ERK), and Wnt/β-catenin, which may synergistically promote stem-progenitor self-renewal and override the inhibitory effect solely incurred from Akt. It is worthy to mention that PI3K signaling can lead to not only Akt-dependent but also Akt–independent activation [51] and the latter might be partly responsible for combating the inhibitory effect from the former.

The present report demonstrates that in spite of exerting tumor-suppressing effects, Akt can render an opposing oncogenic event by resisting cell death induced by the action of cytotoxic drugs. It thereby suggests that not only ectopically expressed Myr-Akt retained authentic functionality, but also that Akt signaling regulates pleotropic downstream substrates, each of which exerts distinct outcomes. For instance, Forkhead transcription factors (FOXO1), BAD, caspase 9, and NF-κB seem to be responsible for protecting cells from apoptosis, whereas mTOR and Wnt/β-catenin signaling might be involved in regulating stem/progenitor cells [23]. In support of this notion, we have observed that, besides repressed EMT and stem cell self-renewal, overexpresssion of Myr-Akt protected cells from apoptotic death induced by Paclitaxel as well as by Doxorubicin (Figure 7). Coincidently, our finding about resistance to apoptosis induced by Doxorubicin intervention can be supported by independent data generated from in vitro knockdown of PTEN in cell culture system [52].

We present a novel paradigm that Akt activation can have dichotomous effects on neoplastic progression. Akt’s intrinsic property of tumor-suppressive effects is demonstrated by repression of EMT, cell motility, and stem/progenitor cell expansion, and that the effects are notably distinct from its tumor-promoting functions that enhance cell survival. While the former would confine the target cells (mainly at non- or low-malignant states) to the local sites, the latter partly contributes to its oncogenic effect. Restrained cells then await additional tumorigenic signals presumably provoked from tumor microenvironmental factors or from additional carcinogenic insults that would alleviate the tumor-suppressive effect of Akt prior to metastatic spread. Meanwhile, epithelial cells would be maintained in a viable state during the course of therapeutic drug treatments.