Hunk negatively regulates c-myc to promote Akt-mediated cell survival and mammary tumorigenesis induced by loss of Pten

Elizabeth S. Yeh; George K. Belka; Ann E. Vernon; Chien-Chung Chen; Jason J. Jung; Lewis A. Chodosh

doi:10.1073/pnas.1217415110

Edited by Alex Toker, Beth Israel Deaconess Medical Center, Boston, MA, and accepted by the Editorial Board February 7, 2013 (received for review October 5, 2012)

Abstract

The protooncogenes Akt and c-myc each positively regulate cell growth and proliferation, but have opposing effects on cell survival. These oncogenes cooperate to promote tumorigenesis, in part because the prosurvival effects of Akt offset the proapoptotic effects of c-myc. Akt’s ability to counterbalance c-myc’s proapoptotic effects has primarily been attributed to Akt-induced stimulation of prosurvival pathways that indirectly antagonize the effects of c-myc. We report a more direct mechanism by which Akt modulates the proapoptotic effects of c-myc. Specifically, we demonstrate that Akt up-regulates the adenosine monophosphate-associated kinase (AMPK)-related protein kinase, Hormonally up-regulated neu-associated kinase (Hunk), which serves as an effector of Akt prosurvival signaling by suppressing c-myc expression in a kinase-dependent manner to levels that are compatible with cell survival. Consequently, Akt pathway activation in the mammary glands of Hunk^−/− mice results in induction of c-myc expression to levels that induce apoptosis. c-myc knockdown rescues the increase in apoptosis induced by Hunk deletion in cells in which Akt has been activated, indicating that repression of c-myc is a principal mechanism by which Hunk mediates the prosurvival effects of Akt. Consistent with this mechanism of action, we find that Hunk is required for c-myc suppression and mammary tumorigenesis induced by phosphatase and tensin homolog (Pten) deletion in mice. Together, our findings establish a prosurvival function for Hunk in tumorigenesis, define an essential mechanism by which Akt suppresses c-myc–induced apoptosis, and identify Hunk as a previously unrecognized link between the Akt and c-myc oncogenic pathways.

Hunk is a sucrose nonfermenting 1 (Snf-1)/AMPK family member that was first identified in the murine mammary gland. The wide range of cellular functions elucidated for the AMPK family of protein kinases suggests that its members may also contribute to cancer development and progression (1). Compatible with this suggestion, Hunk is overexpressed in aggressive subsets of human breast, colon, and ovarian cancers (2). Moreover, studies in Hunk^−/− mice have revealed that Hunk is required for the development of human epidermal growth factor receptor 2 (HER2)/neu-induced mammary tumors (3) as well as the metastasis of mammary tumors induced by c-myc (2). Despite these findings, the intracellular functions of this kinase are poorly understood.

Recently, we demonstrated that Hunk is required for the survival and growth of HER2/neu–driven tumor cells as a consequence of Hunk-dependent regulation of p27^kip1 protein levels and subcellular localization (3). Hunk also reportedly promotes the survival of PC12 neuronal cells, although the underlying mechanism has not been elucidated (4). Additional evidence indicates that Hunk mediates epidermal growth factor receptor (EGFR) and HER2/neu signaling and that Hunk expression is up-regulated by these pathways and by their effector pathways Akt and MAPK (3, 5). Together, these observations suggest that Hunk promotes cell survival and may serve as an effector of growth factor signaling.

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is commonly activated during tumorigenesis and promotes cell growth, proliferation, and survival. Mutations in genes of this pathway, including PIK3CA, AKT, and PTEN, occur in ∼30% of human breast cancers and are associated with resistance to therapy in HER2/neu-amplified breast cancers (6). Additionally, nonmutational silencing and loss of PTEN expression occur in up to ∼40% of human breast cancers (7). Due to the high frequency of mutations in this pathway, identifying critical effectors of Akt signaling has the potential to identify novel opportunities for therapeutic intervention.

One such Akt effector is the protooncogene c-myc, which plays a major role in promoting ribosomal RNA (rRNA) biosynthesis, cell growth, and proliferation (8). Notably, whereas Akt promotes cell survival, high levels of myc sensitize cells to apoptosis (9). Indeed, while myc is oncogenic in its own right, its ability to induce tumors is determined by the context-dependent balance between its proliferative and apoptotic effects. Consequently, myc and Akt cooperate to promote tumorigenesis not only because myc mediates growth-promoting effects of Akt, but also because prosurvival effects of Akt offset myc's proapoptotic effects (10, 11).

To date, the ability of Akt to counterbalance myc’s proapoptotic effects has primarily been attributed to Akt-regulated prosurvival pathways that indirectly antagonize the effects of myc (8). We report here that Akt plays a more direct role in modulating myc’s proapoptotic function. Specifically, we demonstrate that Hunk serves as an intermediate effector of Akt prosurvival signaling by moderating the extent to which Akt up-regulates myc. We find that Akt up-regulates Hunk, which in turn suppresses myc expression to levels that are sufficient for the growth-promoting functions of myc, yet are compatible with cell survival. Consequently, Akt activation in mice lacking Hunk results in super induction of myc expression to levels that induce apoptosis. Consistent with this mechanism of action, mammary tumorigenesis induced by Pten deletion is impaired in Hunk^−/− mice, and myc knockdown rescues the proapoptotic effects of deleting Hunk in cells in which Akt has been activated. Together, our findings establish a prosurvival function for Hunk and define a mechanism by which Akt signaling suppresses myc-induced apoptosis.

Results

Hunk Promotes Cell Survival in the Mammary Gland.

To investigate the effects of Hunk on cell survival, we used the postlactation involuting mammary gland as an in vivo model, because this stage of mammary development is characterized by widespread apoptosis. Mammary glands were collected from Hunk^+/+ and Hunk^−/− female mice at d9 of lactation, when rates of apoptosis are low, and from d 1 through 5 of involution, when rates of apoptosis are high. In agreement with prior findings, mammary glands from day 9 lactating Hunk^−/− mice appeared histologically normal (Fig. S1). However, H&E-stained mammary sections from d 4 and 5 of involution revealed accelerated involution in Hunk^−/− mice (Fig. 1A).

Fig. 1.

Hunk promotes cell survival in the mammary gland. (A) H&E-stained sections of abdominal mammary glands from Hunk^+/+ and Hunk ^−/− mice. (B) Quantification of cleaved caspase-3 IF performed on samples from A. *P < 0.05, **P = 0.005.

Consistent with this, immunofluorescence (IF) staining for cleaved caspase-3 revealed increased rates of apoptosis in Hunk^−/− compared to Hunk^+/+ mammary glands beginning at d4 of involution (Fig. 1B). This suggests that Hunk promotes cell survival in mammary epithelial cells during involution.

Hunk Kinase Activity Is Required for Cell Survival in the Mammary Gland.

To determine if Hunk kinase activity is required for cell survival in the involuting mammary gland, transgenic mice were engineered to conditionally express either WT Hunk (THunk) or a kinase-dead allele of Hunk (TK91M) using a doxycycline (dox)-inducible regulatory system driven by the reverse tetracycline transcriptional activator, rtTA (12) (Fig. S2A). Mouse mammary tumor virus (MMTV)-rtTA;TetO-THunk (MTB/THunk) and MMTV-rtTA;TetO-TK91M (MTB/TK91M) mice were mated at 6 wk of age and administered dox to induce Hunk expression in the mammary gland. At d4 of involution, examination of H&E-stained sections revealed that mammary glands from MTB/THunk mice exhibited delayed involution compared with MTB/TK91M mice, which appeared similar to MTB controls (Fig. S2C). Correspondingly, the mammary glands of involuting MTB/THunk mice displayed decreased staining for cleaved caspase-3 (Fig. S2 B and C), indicating that Hunk overexpression is sufficient to promote cell survival in the involuting mammary gland and that Hunk kinase activity is required for this function.

Hunk Suppresses myc and its Transcriptional Targets in the Mammary Gland.

Overexpression of myc in the mammary gland is known to induce apoptosis (13 ⇓–15), and myc is up-regulated in the mammary gland at d4 of involution (16). Therefore, we examined myc expression levels in Hunk^+/+ and Hunk^−/− mice by quantitative real-time PCR (qRT-PCR). This revealed that myc levels are elevated in the mammary glands of Hunk^−/− mice compared with controls beginning at d4 of involution (Fig. 2A). This, in turn, suggested that the increase in apoptosis observed in involuting mammary glands in Hunk^−/− mice might result from de-repression of myc expression.

Fig. 2.

Hunk suppresses myc expression and activity in the mammary gland. (A) qRT-PCR for c-myc mRNA levels in Hunk^+/+ and Hunk^−/− mammary glands at day 9 lactation and d 2–6 of involution. *P < 0.01, **P = 0.01, ***P < 0.05. (B and C) qRT-PCR for cdc25A (B) and cdc25C (C) mRNA levels in Hunk^+/+ and Hunk^−/− mammary glands at d 4–6 of involution. *P < 0.05, **P = 0.05, ***P < 0.001.

We previously reported that Hunk promotes cell survival in HER2/neu-induced mammary tumors by regulating p27^kip1 (3). However, no alterations in p27^kip1 levels were observed in involuting Hunk^−/− mammary glands (Fig. S3A), suggesting that the mechanisms by which Hunk promotes cell survival may be context-specific.

Cell division cycle 25 A (Cdc25A) is an apoptosis-specific transcriptional target of myc (17). In agreement with Hunk-dependent repression of myc expression, cdc25A levels were elevated in involuting Hunk^−/− mammary glands (Fig. 2B), whereas levels of cdc25C—which is not a myc target (17)—were unaffected (Fig. 2C). Moreover, expression levels of six additional myc transcriptional targets were higher in Hunk^−/− mammary glands, four of which reached statistical significance (Fig. S3B). These findings suggest that Hunk inhibits apoptosis by repressing myc expression and myc transcriptional activity.

Hunk Promotes Cell Survival and Regulates myc Expression in a Kinase-Dependent Manner.

To determine whether Hunk-dependent regulation of myc expression requires Hunk kinase activity, we transduced nontransformed HC11 mouse mammary epithelial cells with WT-Hunk, K91M-Hunk, or an empty vector control (Fig. S4A). To induce apoptosis, cells were serum-deprived for 24 h. Analogous to our in vivo findings, expression of WT Hunk protected cells from apoptosis, as indicated by cleaved caspase-3 staining, whereas K91M-Hunk did not (Fig. S4 B and C). Also consistent with our in vivo findings, WT-Hunk suppressed myc and cdc25A expression levels, whereas levels of myc and cdc25A in K91M-Hunk–expressing cells were similar to those in control cells (Fig. S4 D and E). cdc25C levels were unaffected by expression of WT-Hunk or K91M-Hunk (Fig. S4F).

To extend these findings, we compared myc and cdc25A expression levels in mammary gland fibroblasts (MGFs) derived from Hunk^+/+ and Hunk^−/− mice. We found that Hunk^−/− MGFs exhibit elevated levels of myc and cdc25A compared with Hunk^+/+ MGFs, without alterations in cdc25C expression (Fig. S5A–C). Similarly, levels of c-myc and cdc25A, but not cdc25C, were elevated in Hunk^−/− mouse embryonic fibroblasts (Fig. S6).

To determine whether the suppressive effects of Hunk on cdc25A expression are mediated by myc, we next treated Hunk^−/− MGFs with siRNA to prevent the up-regulation of myc induced by Hunk deletion. qRT-PCR analysis confirmed that myc levels, as well as cdc25A levels, were reduced in Hunk^−/− MGFs transfected with myc siRNA compared with a scrambled siRNA control (Fig. S5 D and E).

We next asked if restoring Hunk expression in Hunk^−/− MGFs would rescue myc expression levels. Hunk^−/− MGFs were transduced with WT-Hunk, K91M-Hunk, or an empty vector control (Fig. S5F). As a positive control, myc was stably introduced into Hunk^+/+ cells (Fig. S5G). Consistent with our prior observations, Hunk^−/− control MGFs exhibited higher levels of myc compared with Hunk^+/+ cells (Fig. S5G). As predicted, readdition of WT Hunk, but not K91M-Hunk, to Hunk^−/− cells down-regulated myc and cdc25A mRNA levels (Fig. S5 G and H). These findings demonstrate that Hunk promotes cell survival and regulates c-myc and cdc25A levels in a kinase-dependent manner.

We next evaluated myc protein levels in Hunk^+/+ and Hunk^−/− MGFs treated with the proteasome inhibitor MG132. Consistent with our previous findings, we observed increased myc protein levels in Hunk^−/− cells as well as increased phosphorylation on S62 of myc (Fig. S5I). To determine whether increased myc mRNA and protein levels resulted from alterations in transcriptional regulation, we treated Hunk^+/+ and Hunk^−/− MGFs with the transcription inhibitor Actinomycin D. This revealed that, while myc mRNA levels were higher in Hunk^−/− MGFs, myc mRNA half-life was not altered by Hunk deletion. These results suggest that the elevation in myc levels observed in Hunk^−/− cells is due to increased transcription, rather than increased mRNA stability (Fig. S5 J and K).

Akt Up-regulates Hunk.

We previously showed that Hunk expression is up-regulated by the EGFR and HER2 signaling pathways (3). Because Akt lies downstream of each of these pathways, we asked whether Hunk expression is dependent upon Akt signaling. Consistent with this possibility, treatment of the MMTV-neu derived tumor cell line, SMF, with either the PI3K inhibitor LY294002 or the Akt inhibitor API-2 led to a reduction in Hunk protein levels (Fig. S7A).

We next asked if Hunk expression is positively regulated by Akt in vivo by inducibly expressing a myristoylated (myr) allele of Akt1 in the mammary gland using a dox-inducible mouse model (12). Because deletion of Akt1 promotes apoptosis and mammary involution, we hypothesized that at least some of the prosurvival effects of Akt1 might be mediated by up-regulation of Hunk. MMTV-rtTA/TetO-Akt1 (MTB/TAkt1) bitransgenic mice were treated with dox for 96 h to induce myr-Akt1 transgene expression. Consistent with our observation that Akt inhibition results in Hunk down-regulation, acute in vivo activation of Akt1 signaling up-regulated Hunk expression (Fig. S7B).

Hunk Suppresses myc Expression and Promotes Cell Survival Following Akt Activation.

To determine whether Hunk mediates the prosurvival effects of Akt1, nulliparous control MTB, MTB/TAkt1;Hunk^+/+, and MTB/TAkt1;Hunk^−/− mice were administered dox for 96 h, and harvested mammary glands were stained for cleaved caspase-3 (Fig. S7C). Although acute Akt1 activation induced a modest increase in apoptosis in MTB/TAkt1;Hunk^+/+ mice compared with MTB controls, Akt1 activation induced a twofold higher rate of apoptosis in Hunk^−/− mice compared with Hunk^+/+ mice (Fig. S7D) that could not be attributed to changes in levels of Akt1 activation or cellular proliferation (Fig. S8). These results suggest that Hunk suppresses apoptosis following Akt1 activation, which further supports a prosurvival role for Hunk.

To determine if Hunk regulates myc expression in the context of Akt1 activation, we examined mammary glands from nulliparous MTB/TAkt1;Hunk^+/+ and MTB/TAkt1;Hunk^−/− mice induced with dox for 96 h. myc expression was elevated in MTB/TAkt1;Hunk^+/+ glands compared with MTB controls, suggesting that Akt1 up-regulates both Hunk and myc (Fig. S7 B and E). Notably, myc expression levels were higher in MTB/TAkt1;Hunk^−/− compared with MTB/TAkt1;Hunk^+/+ mammary glands (Fig. S7E), suggesting that Akt1 activation and Hunk-deletion each up-regulate myc via pathways that are at least partially independent.

Hunk and Akt Synergize to Promote Cell Survival but Exert Opposing Effects on myc Expression.

Because Akt1 promotes cell survival during mammary involution (18, 19), we next asked whether Hunk mediates these prosurvival effects. We performed IF for cleaved caspase-3 on day 4 involuting mammary glands harvested from Hunk^+/+ and Hunk^−/− mice as well as from MTB/TAkt1;Hunk^+/+ and MTB/TAkt1;Hunk^−/− mice in which myr-Akt1 had been induced for 5 d. Supporting our hypothesis that Hunk promotes cell survival following Akt1 activation, apoptosis was suppressed in involuting mammary glands from MTB/TAkt1;Hunk^+/+ compared with MTB/TAkt1;Hunk^−/− mice (Fig. 3 A and B). Consistent with the fact that Akt1 promotes cell survival through multiple effector pathways, apoptosis rates were lower in MTB/TAkt1;Hunk^−/− mammary glands than in Hunk^−/− control mice in which Akt had not been activated (Fig. 3 A and B).

Fig. 3.

Hunk mediates Akt-induced cell survival and represses myc expression. (A) Cleaved caspase-3 IF of abdominal mammary glands derived from Hunk^+/+, Hunk^−/−, MTB/TAkt1;Hunk^+/+, and MTB/TAkt1;Hunk^−/− mice at d4 of involution. (B) Quantification of A. *P = 0.01, **P < 0.01. (C) qRT-PCR for c-myc mRNA levels. *P = 0.01, **P < 0.001. (D) qRT-PCR for Hunk mRNA levels in mammary glands derived from dox-induced MTB and MTB/TAkt1;Hunk^+/+ mice at d4 of involution.

In line with prior observations, both myc and Hunk expression were up-regulated in the involuting mammary glands of dox-induced MTB/TAkt1;Hunk^+/+ mice compared with Hunk^+/+ controls (Fig. 3 C and D). As before, myc mRNA levels were elevated in Hunk^−/− mice compared with WT controls (Fig. 3C), confirming that Hunk represses myc expression.

Notably, myc levels were highest in the involuting mammary glands of dox-induced MTB/TAkt1;Hunk^−/− mice (Fig. 3C), further suggesting that the effects of Akt1 activation and Hunk deletion on myc expression are additive. Moreover, similar to our earlier findings (Fig. S3), levels of myc transcriptional targets were higher in involuting mammary glands from MTB/TAkt1;Hunk^−/− compared with MTB/TAkt1;Hunk^+/+ mice (Fig. S9). Together, our data suggest that Hunk and Akt1 exert opposing effects on myc expression in the mammary gland, but collaborate to promote cell survival.

Hunk Mediates the Prosurvival Effects of Akt by Suppressing myc Expression.

Our observations that Akt up-regulates Hunk, that Hunk mediates prosurvival effects of Akt, and that Hunk represses myc expression raised the possibility that Akt-induced Hunk up-regulation suppresses apoptosis by restraining Akt-induced increases in myc expression. Specifically, we hypothesized that abrogating the up-regulation of myc expression observed in Akt1-expressing Hunk^−/− compared with Hunk^+/+ cells would abrogate the increase in apoptosis associated with Hunk deletion in this context. To test this hypothesis, we generated Hunk^+/+ and Hunk^−/− MGFs stably expressing HA-tagged myr-Akt1 or a vector control (Fig. 4A) as an in vitro model in which myc levels could be manipulated using siRNA. In accordance with our in vivo observations (Fig. 3C), expression of myr-Akt1 in Hunk^+/+ MGFs up-regulated both myc and Hunk (Fig. 4 B, C, and E), Hunk^−/− MGFs expressed higher levels of myc than Hunk^+/+ MGFs (Fig. 4E), and Hunk^−/−;myr-Akt1 cells exhibited the highest levels of myc (Fig. 4E).

Fig. 4.

Hunk mediates the prosurvival effects of Akt by suppressing myc expression. (A) IF for HA tag to detect myr-Akt1 in Hunk^+/+ and Hunk^−/− MGFs stably transduced with either HA-myr-Akt1 or an empty vector control. (A and B) qRT-PCR analysis of myc (B) and Hunk (C) mRNA levels in Hunk^+/+ MGFs transduced with HA-myr-Akt1 or control vector. *P < 0.05. (D) IF for cleaved caspase-3 in cells deprived of serum for 24 h and treated with control or c-myc siRNA. (E) qRT-PCR for c-myc mRNA levels.*P < 0.01, **P = 0.001, ***P < 0.001. (F) Quantification of cleaved caspase-3 IF in (C). *P < 0.05, **P < 0.01, ***P = 0.001.

To determine whether the proapoptotic effect of Hunk deletion in MGFs in which Akt1 has been activated is mediated by myc up-regulation, we used siRNA to knock down myc expression in Hunk^−/− and Hunk^−/−;myr-Akt1 cells to levels comparable to those in WT cells, such that Hunk deletion and Akt1 activation did not result in increased myc levels (Fig. 4E). Cells were then serum-deprived for 24 h and assessed for levels of cleaved caspase-3.

Consistent with our in vivo observations (Fig. 3B), apoptosis rates were higher in serum-deprived Hunk^−/− MGFs compared with Hunk^+/+ MGFs, and Akt1 activation markedly suppressed apoptosis in Hunk WT cells (Fig. 4 D and F). Notably, deletion of Hunk impaired the ability of Akt1 to promote survival (Fig. 4 D and F) and also resulted in superinduction of myc levels (Fig. 4E). Indicative of a causal relationship between elevated myc levels and increased apoptosis, myc knockdown rescued cell survival in serum-deprived Hunk^−/− MGFs as well as Hunk^−/−;myr-Akt1 MGFs to levels comparable with those observed in Hunk^+/+ cells (Fig. 4 D and F). Furthermore, consistent with the similar levels of myc expression achieved in Hunk^−/− and Hunk^−/−;myr-Akt1 MGFs by myc siRNA knockdown, Akt1 activation in Hunk^−/− cells had no effect on apoptosis in the presence of myc knockdown. These findings indicate that Hunk promotes the survival of cells subjected to serum deprivation and does so by suppressing myc expression. Further, our findings confirm that Hunk is a prosurvival effector of Akt and demonstrate that Hunk mediates antiapoptotic effects of Akt in serum-deprived cells by suppressing Akt-induced myc up-regulation.

Hunk Is Required for PI3K-Akt–Induced Mammary Tumorigenesis.

Our observation that Hunk mediates prosurvival effects of Akt1 during mammary gland development and in response to serum deprivation suggested that Hunk might also be required for Akt-induced mammary tumorigenesis. To address this, we asked whether mammary tumor latency was altered in Hunk^−/− mice in which the endogenous PI3K-Akt pathway had been activated. Because PTEN is frequently silenced or deleted in human breast cancers, we selected a conditional mouse Pten knockout model (Pten^co/co) to assess the requirement for Hunk in Akt-induced mammary tumorigenesis.

MMTV-rtTA (MTB) and TetO-Cre (TTC1) transgenic mice, which when mated permit dox-inducible expression of Cre, were interbred with mice bearing conditional Pten alleles in a Hunk^+/+ or Hunk^−/− background. MTB/TTC1;Pten^co/co;Hunk^+/+ and MTB/TTC1;Pten^co/co;Hunk^−/− mice were administered dox to trigger biallelic Pten deletion and activation of the endogenous Akt pathway in the mammary glands of transgenic mice (Fig. 5B).

Fig. 5.

Hunk is required for PI3K/Akt-induced mammary tumorigenesis. (A) myc mRNA levels in mammary glands 4 d following Pten deletion in mammary glands from MTB/TTC1;Pten^co/co;Hunk^+/+ and MTB/TTC1;Pten^co/co;Hunk^−/− mice. *P < 0.05. (B) Western blot analysis demonstrating increased levels of pS478 Akt and myc 14 d following Pten deletion in mammary glands from MTB/TTC1;Pten^co/co;Hunk^+/+ and MTB/TTC1;Pten^co/co;Hunk^−/− mice. (C) IF for cleaved caspase-3 in mammary glands from 14-d induced MTB/TTC1;Pten^co/co;Hunk^+/+ and MTB/TTC1;Pten^co/co;Hunk^−/− mice. *P < 0.001. (D) Mammary tumor-free survival curves comparing MTB/TTC1;Pten^co/co;Hunk^+/+ and MTB/TTC1;Pten^co/co;Hunk^−/− mice treated with 2 mg/mL dox. P < 0.005; HR = 2.7.

As predicted based on our findings in inducible Akt1 transgenic mice, myc mRNA and protein levels were up-regulated in dox-induced MTB/TTC1;Pten^co/co;Hunk^+/+ mice compared with non–Pten-deleted MTB controls and were even higher in dox-induced MTB/TTC1;Pten^co/co;Hunk ^−/− mice (Fig. 5 A and B). Consistent with this, expression of myc target genes were also higher in mammary glands derived from MTB/TTC1;Pten^co/co;Hunk^−/− compared with MTB/TTC1;Pten^co/co; Hunk^+/+ controls (Fig. S10A). Rates of apoptosis paralleled these alterations in myc expression because dox-induced MTB/TTC1;Pten^co/co;Hunk^−/− mice exhibited substantially higher rates of apoptosis than dox-induced MTB/TTC1;Pten^co/co;Hunk^+/+ controls (Fig. 5C).

Consistent with this increase in apoptosis, mammary tumor development was markedly delayed in MTB/TTC1;Pten^co/co;Hunk^−/− mice, which exhibited a median tumor latency of 170 d compared with 77 d for MTB/TTC1;Pten^co/co;Hunk^+/+ controls (Fig. 5D). In aggregate, our results indicate that Hunk-mediated suppression of Akt-induced myc expression is required for the prosurvival effects of Akt and demonstrate that Hunk is required for mammary tumorigenesis induced by pathophysiologically relevant levels of PI3K-Akt activation.

Discussion

Myc exerts opposing effects on tumorigenesis whereby it positively regulates cell growth and proliferation, while promoting cell death when expressed at high levels (8). Accordingly, myc expression is tightly regulated. Although Akt induces myc expression and high levels of myc are proapoptotic, the net effect of Akt activation is to promote cell survival. The ability of Akt signaling to counteract myc-induced apoptosis, and thereby synergize with myc in tumorigenesis, has largely been attributed to Akt’s ability to regulate multiple cell survival pathways (8, 20). We now identify a direct mechanism by which Akt suppresses the proapoptotic effects of myc in which Akt up-regulates myc while simultaneously activating a Hunk kinase–dependent pathway that acts to repress myc expression below a critical threshold at which this protooncogene would induce cell death.

Our findings indicate that Hunk acts as a prosurvival effector of Akt by restraining the magnitude of Akt-induced myc up-regulation, thereby blunting myc-induced apoptosis. Using in vitro and in vivo Hunk-deficient models, we demonstrate that Akt simultaneously up-regulates Hunk and myc expression and that Hunk, in turn, moderates the induction of myc by Akt, thereby preventing myc-induced apoptosis. Thus, in the presence of Hunk, Akt activation has a net prosurvival effect, whereas in the absence of Hunk this balance is shifted toward cell death. These findings identify Hunk as a previously unrecognized link between the Akt and myc oncogenic pathways.

Although Hunk has previously been implicated in mammary tumorigenesis as well as metastasis, a physiological role for this kinase has not been defined (2, 3, 5). We now demonstrate that Hunk maintains the balance between cell survival and cell death during mammary gland development by suppressing the up-regulation of myc that normally occurs during postlactational involution. This physiological function parallels the pathophysiological effects of Hunk in oncogenic contexts in which the Akt pathway has been activated.

Evidence to date indicates that Hunk is expressed at high levels in HER2/neu-amplified human breast cancers, is up-regulated by the EGFR and HER2/neu pathways, and is required for efficient EGFR and HER2/neu signaling (2, 3). These findings suggest that Hunk may play an important role in mediating growth factor signaling and tumorigenesis.

Notably, despite the fact that Akt is downstream of HER2/neu, the mechanisms by which Hunk mediates the prosurvival effects of Akt and HER2/neu signaling differ, the former being attributed to suppression of myc expression, and the latter to regulation of p27^kip1. This indicates that Hunk exerts prosurvival effects via multiple pathways whose relative importance may differ between cells that have activated Akt versus HER2/neu. This model would be consistent with the finding that PIK3CA mutations occur in up to one-quarter of HER2/neu-amplified human breast cancers, despite the fact that HER2/neu amplification is strongly associated with activation of Akt signaling (6). This suggests that primary activation of PI3K/Akt signaling may have tumor-promoting qualities distinct from those resulting from downstream activation of Akt by HER2/neu.

Activation of Akt, either by PIK3CA mutation, HER2/neu amplification, or loss of PTEN, is one of the most commonly occurring alterations in human breast cancer (21). Therefore, identifying essential effectors of Akt signaling may reveal novel opportunities for therapeutic intervention. Toward this end, using Akt1 transgenic and Pten-conditional knockout mouse models, we have demonstrated that Hunk is up-regulated by Akt and that Hunk is required for the survival of cells in which PI3K-Akt signaling has been activated. Consistent with this, we have found that Hunk is required for mammary tumorigenesis induced by Pten deletion in genetically engineered mice. This observation extends our previous finding that Hunk is required for the development of HER2/neu-induced mammary tumors (3), which exhibit a luminal epithelial phenotype, by demonstrating that Hunk is also required for the development of mammary tumors in Pten mutant mice, which display basal-like characteristics (22).

This latter finding is of particular interest in light of a recent report proposing that Hunk functions as a suppressor of metastasis, based on experiments in basal-like human breast cancer cell lines (23). To extend their in vitro findings, these authors suggested that crossing Hunk^−/− mice to mouse models for HER2/neu as well as basal-like breast cancers would yield information pertinent to human breast cancer. Indeed, by means of such crosses, we have determined that Hunk is required for the development of both luminal (i.e., HER2/neu-induced) and basal-like (i.e., Pten deletion–induced) mammary cancers, but does not suppress metastasis in either model (Fig. S10B) (3). As such, our findings are consistent with a protumorigenic, prosurvival function for Hunk that is analogous to its physiological role in the involuting mammary gland, but do not provide evidence to support a role for Hunk in suppressing metastasis. Indeed, the observation that Hunk suppresses metastasis in xenograft models of human basal-like breast cancer cell lines is directly at odds with our in vivo finding that Hunk is required for mammary tumor metastasis in MMTV-c-myc mice (2).

Beyond cell survival, Akt functions as a critical regulator of cell growth, protein synthesis, and metabolism (24). In light of our demonstration that Hunk serves as a link between the Akt and myc oncogenic pathways, the possibility that Hunk may regulate cell growth and/or metabolism is intriguing. In this regard, we have found that the ability of Hunk to promote cell survival in vitro is most pronounced under serum-limiting conditions, raising the possibility of a nutrient-sensing capacity for this AMPK-related kinase (1). Indeed, both Akt and AMPK converge on the mammalian target of rapamycin signaling pathway (20, 25). Consequently, it will be interesting to determine whether Hunk has a role in this central metabolic signaling pathway.

In summary, we have identified a mechanism mediated by the AMPK-related protein kinase Hunk by which Akt opposes myc-induced apoptosis. Our findings reveal that Akt up-regulates Hunk, which in turn suppresses Akt-induced up-regulation of myc, as well as apoptosis, in a Hunk kinase–dependent manner. Our findings further demonstrate that this regulatory interaction plays an essential role in mammary tumorigenesis induced by deletion of the Pten tumor suppressor. As such, targeting Hunk kinase activity may have beneficial therapeutic effects in human cancers.

Materials and Methods

Cell Culture.

Cell lines were grown as described (26). MGFs were isolated by manual disruption and trypsinization of mammary tissue and immortalized by introduction of CMV-p53_DD (Vassiliki Karantza, University of Medicine and Dentistry, New Brunswick, NJ), via electroporation (0.22 kV; 950 uF). For media formulations, see SI Materials and Methods.

siRNAs were purchased from Qiagen (see SI Materials and Methods for sequences). Transfections were carried out in Dulbecco's Modified Eagle Medium without serum using Lipofectamine 2000 (Invitrogen). Cells were maintained for 8 h before readdition of media containing 2.5% (vol/vol) Super Calf Serum (GemCell) for 18 h before collection. Cells were maintained in media without serum for 24 h before cell survival analysis. Retroviral transductions were carried out as described (2). LY294002 and API-2 were purchased from Sigma and Berry and Associates, respectively.

Animal and Tissue Preparation.

Mice were housed in accordance with University of Pennsylvania Institutional Animal Care and Use Committee guidelines. Tumorigenesis and histological analysis were performed as described (3). Pten conditional knockout mice were obtained from Jackson Labs.

Immunoblotting and IF.

Protein lysates were prepared in lysis buffer (SI Materials and Methods) with HALT inhibitor mixture (Thermo Scientific). Bound antibodies (SI Materials and Methods) were detected with ECL (Amersham). IF staining was performed as described (3).

RNA Isolation and qRT-PCR.

RNA isolation (RNeasy kit, Qiagen) and RT reactions (Advantage cDNA High Capacity Kit, ABI) were performed per manufacturers’ instructions. qRT-PCR was performed on the ABI 7900 HT Fast Realtime PCR system using 6-carboxyfluorescein labeled Taqman probes (ABI). mRNA levels were normalized to TATA binding protein.

Statistical Analysis.

Student t test was used for statistical analysis. Error bars represent the SEM. For in vivo experiments, at least three mice per genotype were evaluated. For in vitro experiments, data represent at least two experimental repeats that used at least three samples per experiment.

Acknowledgments

This work was supported by grants from the National Cancer Institute (to L.A.C.), Department of Defense Breast Cancer Research Program (to L.A.C. and E.S.Y.), and the Breast Cancer Research Foundation (to L.A.C.).

Footnotes

↵¹To whom correspondence should be addressed. E-mail: chodosh{at}mail.med.upenn.edu.

Author contributions: E.S.Y. and L.A.C. designed research; E.S.Y. and G.K.B. performed research; E.S.Y., A.E.V., C.-C.C., and J.J.J. contributed new reagents/analytic tools; E.S.Y. and L.A.C. analyzed data; and E.S.Y. and L.A.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. A.T. is a guest editor invited by the Editorial Board.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1217415110/-/DCSupplemental.

Freely available online through the PNAS open access option.

References

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1. Bright NJ,
2. Thornton C,
3. Carling D
(2009) The regulation and function of mammalian AMPK-related kinases. Acta Physiol (Oxf) 196(1):15–26.
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1. Wertheim GB,
2. et al.
(2009) The Snf1-related kinase, Hunk, is essential for mammary tumor metastasis. Proc Natl Acad Sci USA 106(37):15855–15860.
OpenUrl Abstract/FREE Full Text
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1. Yeh ES,
2. et al.
(2011) Hunk is required for HER2/neu-induced mammary tumorigenesis. J Clin Invest 121(3):866–879.
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1. Korobko IV,
2. et al.
(2010) Pro-survival activity of the MAK-V protein kinase in PC12 cells. Cell Cycle 9(20):4248–4249.
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1. Komurov K,
2. et al.
(2010) Comprehensive mapping of the human kinome to epidermal growth factor receptor signaling. J Biol Chem 285(27):21134–21142.
OpenUrl Abstract/FREE Full Text
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1. Baselga J
(2011) Targeting the phosphoinositide-3 (PI3) kinase pathway in breast cancer. Oncologist 16(Suppl 1):12–19.
OpenUrl Abstract/FREE Full Text
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1. Hollander MC,
2. Blumenthal GM,
3. Dennis PA
(2011) PTEN loss in the continuum of common cancers, rare syndromes and mouse models. Nat Rev Cancer 11(4):289–301.
OpenUrl CrossRef PubMed
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1. Pelengaris S,
2. Khan M,
3. Evan G
(2002) c-MYC: More than just a matter of life and death. Nat Rev Cancer 2(10):764–776.
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1. Evan GI,
2. et al.
(1992) Induction of apoptosis in fibroblasts by c-myc protein. Cell 69(1):119–128.
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1. Clegg NJ,
2. et al.
(2011) MYC cooperates with AKT in prostate tumorigenesis and alters sensitivity to mTOR inhibitors. PLoS ONE 6(3):e17449.
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1. Chan JC,
2. et al.
(2011) AKT promotes rRNA synthesis and cooperates with c-MYC to stimulate ribosome biogenesis in cancer. Sci Signal 4(188):ra56.
OpenUrl Abstract/FREE Full Text
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1. Gunther EJ,
2. et al.
(2002) A novel doxycycline-inducible system for the transgenic analysis of mammary gland biology. FASEB J 16(3):283–292.
OpenUrl Abstract/FREE Full Text
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1. Blakely CM,
2. et al.
(2005) Developmental stage determines the effects of MYC in the mammary epithelium. Development 132(5):1147–1160.
OpenUrl Abstract/FREE Full Text
↵
1. Sutherland KD,
2. et al.
(2006) c-myc as a mediator of accelerated apoptosis and involution in mammary glands lacking Socs3. EMBO J 25(24):5805–5815.
OpenUrl CrossRef PubMed
↵
1. Wang X,
2. et al.
(2011) Phosphorylation regulates c-Myc’s oncogenic activity in the mammary gland. Cancer Res 71(3):925–936.
OpenUrl Abstract/FREE Full Text
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1. Strange R,
2. Li F,
3. Saurer S,
4. Burkhardt A,
5. Friis RR
(1992) Apoptotic cell death and tissue remodelling during mouse mammary gland involution. Development 115(1):49–58.
OpenUrl Abstract
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1. Galaktionov K,
2. Chen X,
3. Beach D
(1996) Cdc25 cell-cycle phosphatase as a target of c-myc. Nature 382(6591):511–517.
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1. Maroulakou IG,
2. et al.
(2008) Distinct roles of the three Akt isoforms in lactogenic differentiation and involution. J Cell Physiol 217(2):468–477.
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1. Schwertfeger KL,
2. Richert MM,
3. Anderson SM
(2001) Mammary gland involution is delayed by activated Akt in transgenic mice. Mol Endocrinol 15(6):867–881.
OpenUrl Abstract/FREE Full Text
↵
1. Sekulić A,
2. et al.
(2000) A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res 60(13):3504–3513.
OpenUrl Abstract/FREE Full Text
↵
1. Wickenden JA,
2. Watson CJ
(2010) Key signalling nodes in mammary gland development and cancer. Signalling downstream of PI3 kinase in mammary epithelium: A play in 3 Akts. Breast Cancer Res 12(2):202.
OpenUrl CrossRef PubMed
↵
1. Li G,
2. et al.
(2002) Conditional loss of PTEN leads to precocious development and neoplasia in the mammary gland. Development 129(17):4159–4170.
OpenUrl Abstract/FREE Full Text
↵
1. Quintela-Fandino M,
2. et al.
(2010) HUNK suppresses metastasis of basal type breast cancers by disrupting the interaction between PP2A and cofilin-1. Proc Natl Acad Sci USA 107(6):2622–2627.
OpenUrl Abstract/FREE Full Text
↵
1. Plas DR,
2. Thompson CB
(2005) Akt-dependent transformation: There is more to growth than just surviving. Oncogene 24(50):7435–7442.
OpenUrl CrossRef PubMed
↵
1. Shaw RJ,
2. et al.
(2004) The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6(1):91–99.
OpenUrl CrossRef PubMed
↵
1. Morrison BW,
2. Leder P
(1994) neu and ras initiate murine mammary tumors that share genetic markers generally absent in c-myc and int-2-initiated tumors. Oncogene 9(12):3417–3426.
OpenUrl PubMed

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Proceedings of the National Academy of Sciences: 110 (15)

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Submit

[1] ↵
Bright NJ,
Thornton C,
Carling D
(2009) The regulation and function of mammalian AMPK-related kinases. Acta Physiol (Oxf) 196(1):15–26.
OpenUrl CrossRef PubMed

[2] Bright NJ,

[3] Thornton C,

[4] Carling D

[5] ↵
Wertheim GB,
et al.
(2009) The Snf1-related kinase, Hunk, is essential for mammary tumor metastasis. Proc Natl Acad Sci USA 106(37):15855–15860.
OpenUrl Abstract/FREE Full Text

[6] Wertheim GB,

[7] et al.

[8] ↵
Yeh ES,
et al.
(2011) Hunk is required for HER2/neu-induced mammary tumorigenesis. J Clin Invest 121(3):866–879.
OpenUrl CrossRef PubMed

[9] Yeh ES,

[10] et al.

[11] ↵
Korobko IV,
et al.
(2010) Pro-survival activity of the MAK-V protein kinase in PC12 cells. Cell Cycle 9(20):4248–4249.
OpenUrl CrossRef PubMed

[12] Korobko IV,

[13] et al.

[14] ↵
Komurov K,
et al.
(2010) Comprehensive mapping of the human kinome to epidermal growth factor receptor signaling. J Biol Chem 285(27):21134–21142.
OpenUrl Abstract/FREE Full Text

[15] Komurov K,

[16] et al.

[17] ↵
Baselga J
(2011) Targeting the phosphoinositide-3 (PI3) kinase pathway in breast cancer. Oncologist 16(Suppl 1):12–19.
OpenUrl Abstract/FREE Full Text

[18] Baselga J

[19] ↵
Hollander MC,
Blumenthal GM,
Dennis PA
(2011) PTEN loss in the continuum of common cancers, rare syndromes and mouse models. Nat Rev Cancer 11(4):289–301.
OpenUrl CrossRef PubMed

[20] Hollander MC,

[21] Blumenthal GM,

[22] Dennis PA

[23] ↵
Pelengaris S,
Khan M,
Evan G
(2002) c-MYC: More than just a matter of life and death. Nat Rev Cancer 2(10):764–776.
OpenUrl CrossRef PubMed

[24] Pelengaris S,

[25] Khan M,

[26] Evan G

[27] ↵
Evan GI,
et al.
(1992) Induction of apoptosis in fibroblasts by c-myc protein. Cell 69(1):119–128.
OpenUrl CrossRef PubMed

[28] Evan GI,

[29] et al.

[30] ↵
Clegg NJ,
et al.
(2011) MYC cooperates with AKT in prostate tumorigenesis and alters sensitivity to mTOR inhibitors. PLoS ONE 6(3):e17449.
OpenUrl CrossRef PubMed

[31] Clegg NJ,

[32] et al.

[33] ↵
Chan JC,
et al.
(2011) AKT promotes rRNA synthesis and cooperates with c-MYC to stimulate ribosome biogenesis in cancer. Sci Signal 4(188):ra56.
OpenUrl Abstract/FREE Full Text

[34] Chan JC,

[35] et al.

[36] ↵
Gunther EJ,
et al.
(2002) A novel doxycycline-inducible system for the transgenic analysis of mammary gland biology. FASEB J 16(3):283–292.
OpenUrl Abstract/FREE Full Text

[37] Gunther EJ,

[38] et al.

[39] ↵
Blakely CM,
et al.
(2005) Developmental stage determines the effects of MYC in the mammary epithelium. Development 132(5):1147–1160.
OpenUrl Abstract/FREE Full Text

[40] Blakely CM,

[41] et al.

[42] ↵
Sutherland KD,
et al.
(2006) c-myc as a mediator of accelerated apoptosis and involution in mammary glands lacking Socs3. EMBO J 25(24):5805–5815.
OpenUrl CrossRef PubMed

[43] Sutherland KD,

[44] et al.

[45] ↵
Wang X,
et al.
(2011) Phosphorylation regulates c-Myc’s oncogenic activity in the mammary gland. Cancer Res 71(3):925–936.
OpenUrl Abstract/FREE Full Text

[46] Wang X,

[47] et al.

[48] ↵
Strange R,
Li F,
Saurer S,
Burkhardt A,
Friis RR
(1992) Apoptotic cell death and tissue remodelling during mouse mammary gland involution. Development 115(1):49–58.
OpenUrl Abstract

[49] Strange R,

[50] Li F,

[51] Saurer S,

[52] Burkhardt A,

[53] Friis RR

[54] ↵
Galaktionov K,
Chen X,
Beach D
(1996) Cdc25 cell-cycle phosphatase as a target of c-myc. Nature 382(6591):511–517.
OpenUrl CrossRef PubMed

[55] Galaktionov K,

[56] Chen X,

[57] Beach D

[58] ↵
Maroulakou IG,
et al.
(2008) Distinct roles of the three Akt isoforms in lactogenic differentiation and involution. J Cell Physiol 217(2):468–477.
OpenUrl CrossRef PubMed

[59] Maroulakou IG,

[60] et al.

[61] ↵
Schwertfeger KL,
Richert MM,
Anderson SM
(2001) Mammary gland involution is delayed by activated Akt in transgenic mice. Mol Endocrinol 15(6):867–881.
OpenUrl Abstract/FREE Full Text

[62] Schwertfeger KL,

[63] Richert MM,

[64] Anderson SM

[65] ↵
Sekulić A,
et al.
(2000) A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res 60(13):3504–3513.
OpenUrl Abstract/FREE Full Text

[66] Sekulić A,

[67] et al.

[68] ↵
Wickenden JA,
Watson CJ
(2010) Key signalling nodes in mammary gland development and cancer. Signalling downstream of PI3 kinase in mammary epithelium: A play in 3 Akts. Breast Cancer Res 12(2):202.
OpenUrl CrossRef PubMed

[69] Wickenden JA,

[70] Watson CJ

[71] ↵
Li G,
et al.
(2002) Conditional loss of PTEN leads to precocious development and neoplasia in the mammary gland. Development 129(17):4159–4170.
OpenUrl Abstract/FREE Full Text

[72] Li G,

[73] et al.

[74] ↵
Quintela-Fandino M,
et al.
(2010) HUNK suppresses metastasis of basal type breast cancers by disrupting the interaction between PP2A and cofilin-1. Proc Natl Acad Sci USA 107(6):2622–2627.
OpenUrl Abstract/FREE Full Text

[75] Quintela-Fandino M,

[76] et al.

[77] ↵
Plas DR,
Thompson CB
(2005) Akt-dependent transformation: There is more to growth than just surviving. Oncogene 24(50):7435–7442.
OpenUrl CrossRef PubMed

[78] Plas DR,

[79] Thompson CB

[80] ↵
Shaw RJ,
et al.
(2004) The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6(1):91–99.
OpenUrl CrossRef PubMed

[81] Shaw RJ,

[82] et al.

[83] ↵
Morrison BW,
Leder P
(1994) neu and ras initiate murine mammary tumors that share genetic markers generally absent in c-myc and int-2-initiated tumors. Oncogene 9(12):3417–3426.
OpenUrl PubMed

[84] Morrison BW,

[85] Leder P

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