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. Author manuscript; available in PMC: 2016 Feb 10.
Published in final edited form as: Cancer Cell. 2015 Oct 12;28(4):472–485. doi: 10.1016/j.ccell.2015.09.005

An Epigenetic Pathway Regulates Sensitivity of Breast Cancer Cells to HER2 Inhibition via FOXO/c-Myc Axis

Smita Matkar 1, Paras Sharma 1, Shubin Gao 3, Buddha Gurung 1, Bryson W Katona 1, Jennifer Liao 1, Abdul Bari Muhammad 1, Xiang-Cheng Kong 1, Lei Wang 1,4, Guanghui Jin 3, Chi Dang 2, Xianxin Hua 1,§
PMCID: PMC4748715  NIHMSID: NIHMS751692  PMID: 26461093

SUMMARY

Human epidermal growth factor receptor 2 (HER2) is upregulated in a subset of human breast cancers. However, the cancer cells often quickly develop an adaptive response to HER2 kinase inhibitors. We found that an epigenetic pathway involving MLL2 is crucial for growth of HER2+ cells and MLL2 reduces sensitivity of the cancer cells to a HER2 inhibitor, Lapatinib. Lapatinib-induced FOXO transcription factors, normally tumor-suppressing, paradoxically upregulate c-Myc epigenetically, in concert with a cascade of MLL2-associating epigenetic regulators, to dampen sensitivity of the cancer cells to Lapatinib. An epigenetic inhibitor suppressing c-Myc synergizes with Lapatinib to suppress cancer growth in vivo, partly by repressing the FOXO/c-Myc axis, unraveling an epigenetically regulated FOXO/c-Myc axis as a potential target to improve therapy.

INTRODUCTION

The gene for human epidermal growth factor receptor 2 (HER2 or ERBB2) is often amplified or over-expressed in a subgroup of breast cancers and is associated with poor prognosis (Slamon et al., 1987). The HER2/EGFR (Epidermal Growth Factor Receptor) complex can activate a downstream cascade of protein kinases including PI3K and AKT, promoting tumorigenesis by increasing cell proliferation and survival (Schlessinger, 2000). Trastuzumab (Herceptin), a monoclonal antibody against the ectodomain of HER2, and the small molecule HER2 tyrosine kinase inhibitor Lapatinib (Tykerb) are approved for treating HER2-overexpressing breast cancer (Molina et al., 2001; Wood et al., 2004). While Trastuzumab resistance is associated with increased signaling from the insulin-like growth factor-I receptor (IGF-IR) or a PTEN mutation (Lu et al., 2001; Nagata et al., 2004), Lapatinib exerts its antitumor activity in a PTEN-independent manner (Xia et al., 2007), and thus remains useful for treating certain patients with resistance to Herceptin. Adaptive response to Lapatinib or PI3K/AKT inhibitor can develop via several means including increased estrogen receptor signaling and upregulation of pro-survival factors and receptor tyrosine kinases (such as HER3 and IGF-1R) (Chandarlapaty et al., 2011; Garrett et al., 2011; Muranen et al., 2011; Xia et al., 2006). However, precise mechanisms underlying the epigenetic response to Lapatinib in terms of controlling sensitivity or resistance are poorly understood. As such, understanding whether/how sensitivity to Lapatinib is epigenetically or transcriptionally controlled in HER2+ cancer, and developing more effective treatment modalities based on these mechanisms are highly desirable.

The HER2 pathway can activate various downstream effectors including PI3K and AKT, which phosphorylates FOXO transcription factors and results in their subsequent sequestration in the cytoplasm, suppressing their transcriptional activity (Greer and Brunet, 2005). FOXOs are known as tumor suppressors as they upregulate cyclin dependent kinase inhibitors (CDKIs) p21Cip1 and p27Kip1 and pro-apoptotic proteins like Bim (Eijkelenboom and Burgering, 2013; Greer and Brunet, 2005). Inhibiting the kinase activity of HER2 or PI3K/AKT by cognate kinase inhibitors leads to reduction in phosphorylation of FOXOs, and thus triggers their translocation into the nucleus and up-regulates transcription of their target genes (Gilley et al., 2003). An early response to treatment of breast cancer cells with PI3K/AKT inhibitors or Lapatinib is enhanced translocation of FOXOs into the nucleus, leading to up-regulation of several receptor tyrosine kinases (RTKs) and resistance to the inhibitors (Chandarlapaty et al., 2011; Garrett et al., 2011; Muranen et al., 2011).

Targeting epigenetically-regulated pathways in cancer cells is a rapidly emerging approach for therapy (Yoo and Jones, 2006). Epigenetic landscapes are commonly altered in cancer cells, and the promise of therapeutically targeting such pathways in breast cancers has been limited due to a poor understanding of the epigenetic regulatory mechanisms in response to the HER2/AKT inhibitors. Mixed lineage leukemia (MLL) proteins contain a highly conserved SET domain that catalyzes histone 3 lysine 4 (H3K4) trimethylation (Krivtsov and Armstrong, 2007), which is involved in the up-regulation of gene transcription (Gu et al., 1992). In humans, there are several proteins in the MLL family such as MLL (KMT2A), MLL2 (KMT2D), MLL3 (KMT2C), and MLL4 (KMT2B), and MLL and MLL2 associate with co-factors WDR5, RBBP5 and ASH2L. However, it is unclear whether any member of the MLL family is involved in regulating sensitivity to HER2 inhibitors. Thus it is important to investigate whether epigenetic regulation cross-talks with the HER2/AKT pathway to regulate breast cancer cell signaling and sensitivity to HER2/AKT inhibitors. In our current study, we investigated the potential crosstalk between epigenetic pathways and the sensitivity to Lapatinib in HER2+ breast cancers.

RESULTS

shRNA-based screening identified multiple components in MLL complexes that are crucial for growth of BT474 breast cancer cells

To determine the potential impact of the epigenetic modulators on regulating sensitivity/resistance of HER2+ breast cancer cells to targeted drugs, we chose a two-tier approach—first identifying the epigenetic regulators that are crucial for cancer cell growth, followed by examining the potential role of the identified epigenetic modulators in regulating sensitivity to a HER2-inhibition drug such as Lapatinib. To this end, first we assembled an shRNA library targeting epigenetic regulators including multiple histone methyl- or acetyl-transferases. Four to six distinct shRNAs targeting each gene product were packaged in recombinant lentiviruses and were transduced into HER2+ BT474 cells in 96 well plates (Figure 1A). We monitored growth of the resulting puromycin-resistant cells and found that knockdown of multiple epigenetic regulators led to reduced growth of the BT474 cells (Table S1). Notably, knocking down various components of the MLL (MLL and MLL2 share multiple components, or COMPASS), including WDR5, ASH2L, and RBBP5 (Issaeva et al., 2007; Smith et al., 2011), decreased the growth of BT474 cells (Figure 1B). In contrast, shRNAs targeting MLL3 and MLL4, two homologous methyltransferases (Figure S1A), failed to repress cell growth in either the original screen or in verified knockdown experiments (Table S1 and data not shown).

Figure 1. shRNA-based screening identified multiple components in MLL complexes that are crucial for growth of BT474 breast cancer cells and expression of c-Myc and BCL2.

Figure 1

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(A) A Schema for screening shRNA library in BT474 cells. (B) Summary of the impact of knockdown of each of the MLL or MLL2 components on cell growth in the screening. (C,D) Impact of knockdown of MLL (C) or MLL2 (D) on cell growth in large dishes of BT474 cells. (E,F) Impact of knockdown of MLL (E) or MLL2 (F) on expression of MLL or MLL2 and Bclxl, Bcl2 and c-Myc using qRT-PCR assay. Error bars indicate +/− SD. Also see Figure S1 and Table S1.

We further verified the impact of knockdown of MLL, MLL2 and their co-factors on cell growth through dedicated shRNA experiments. To do this, we transduced control or MLL or MLL2 shRNA-expressing recombinant viruses to BT474 cells, and found that knockdown of either MLL or MLL2 reduced the cell growth (Figure 1C-D). We also knocked down other components of MLL2 complex including WDR5 and ASH2L, and BPTF, a reader of H3K4 trimethylated by MLLs, as well as histone acetyltransferase GCN5, and found that their knockdown resulted in reduction of cell number (Figure S1B). These results suggest that MLL and MLL2 and their related factors play an important role in growth of HER2 + BT474 cells.

MLL and MLL2 regulate distinct sets of genes in HER2+ breast cancer cells

Recent work indicates that Bcl2 family proteins play a crucial role in tumorigenesis and chemoresistance of breast cancer cells and c-Myc is important in regulating survival and proliferation of breast cancer cells (Dawson et al., 2010; Lang et al., 2011; Liao and Dickson, 2000; Vaillant et al., 2013). As such we determined whether MLL and MLL2 are crucial for their expression. We found that shRNA-mediated knockdown of MLL decreased the expression of Bclxl but not Bcl2 or c-Myc (Figure 1E). In contrast, MLL2 knockdown had no effect on Bclxl expression, but decreased the expression of Bcl2 and c-Myc (Figure 1F). Collectively, these results indicate that MLL2, but not MLL, is crucial for expression of c-Myc and Bcl2 in the BT474 breast cancer cells.

HER2 inhibitor induces expression of c-Myc and maintains the HER2+ breast cancer cells at a steady state

Activation of Ras/HER2/PI3K signaling pathways alters the expression of c-Myc via affecting transcription, mRNA translation, and protein stability (Galmozzi et al., 2004; Sears et al., 2000; Zhu et al., 2008). Bcl2 is also upregulated in the HER2+ breast cancer cells treated with Lapatinib (Xia et al., 2006), but the impact of Lapatinib on c-Myc was not determined. We sought to determine whether MLL2-regulated c-Myc is influenced by the highly activated HER2 signaling pathway, before we set out to understand MLL2-mediated regulation of c-Myc expression. We found that Lapatinib induced Bcl2 mRNA levels in BT474 cells as expected (Figure 2A-B). Notably, Lapatinib also increased mRNA level of c-Myc (Figure 2A). FOXO transcription factors are phosphorylated by AKT and inhibition of AKT-mediated phosphorylation of FOXOs activates FOXOs by increasing their localization in the nucleus. We found that Lapatinib treatment increased c-Myc mRNA and FOXO1 mRNA after 8 hr of treatment, an effect lasting for several days on daily treatment (Figure S2A-C).

Figure 2. Effect of Lapatinib on expression of c-Myc and Bcl2, both upregulated by MLL2 and HER2 inhibitor Lapatinib.

Figure 2

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(A) BT474 cells were treated with Lapatinib (200 nM) for 3 days, followed by additional 18 hr, prior to qRT-PCR analysis for c-Myc and Bcl2. (B) BT474 cells were treated with DMSO or Lapatinib (200 nM) for the indicated days, collected 18 hr after the last treatment for Western blot analysis with the indicated antibodies. (C, D) In experiments parallel to (B), the dead cells (C) and viable cells (D) were counted on the indicated days. (E, F) MDA-MB-361, were treated with 50 nM AKTi, MK2206, for 4 days, collected 18 hr after the last treatment for analysis for expression of c-Myc and Bcl2 at the mRNA level using qRT-PCR (E) or protein levels of various proteins using Western blotting (F). Error bars indicate +/− SD. Also see Figure S2.

Lapatinib treatment decreased c-Myc protein level on day 1 of treatment (data not shown). However, daily treatment gradually increased the protein level of c-Myc (Figure 2B), and consistently, reduced phosphorylation of AKT and increased protein level of HER3 (Figure 2B), a protein known to be up-regulated by Lapatinib (Chandarlapaty et al., 2011; Garrett et al., 2011; Lu et al., 2001). But Lapatinib increased expression of a pro-apototic protein, Bim (Figure 2B). Moreover, Lapatinib also reduced phosphorylation of FOXO1 (Figure 2B), whose phosphorylation by active AKT leads its sequestration in the cytoplasm and inactivation (Gilley et al., 2003), but increased the total FOXO1 (Figure 2B).

If Lapatinib induces c-Myc expression via suppressing AKT-mediated FOXO phosphorylation, then, treating BT474 cells with an AKT inhibitor should also lead to increased expression of c-Myc. Indeed, treatment of BT474 cells with an AKT inhibitor, MK2206 (referred to as AKTi hereafter), increased the level of c-Myc at RNA and protein level, but reduced phosphorylation of AKT and FOXO1 (Figure S2D and data not shown). Altogether, these results suggest that Lapatinib and AKTi led to suppression of the HER2/PI3K/AKT pathway, reducing AKT activity and consequently up-regulating c-Myc and FOXO1.

We found that the cell death occurred rapidly after Lapatinib treatment for the first three days and thereafter the number of the dead cells decreased (Figure 2C). However, while control cells continued to grow, even at day 6 following the treatment, Lapatinib did not substantially reduce the number of the viable cells as compared to the cell number at the start of the Lapatinib treatment (Figure 2D). Rather, during this period, Lapatinib led to an overall steady state in viable cell number. Careful monitoring of cell cycle showed that Lapatinib reduced BrdU uptake at S-phase of the cancer cells from 18.8% of cells to 2.32%, a lower yet still detectable level (Figure S2E). These findings suggest that Lapatinib-treated cells reached equilibrium of cell division and death, displaying loss of sensitivity or resistance to treatment by Lapatinib.

AKT inhibitor induces c-Myc expression in breast cancer cells with enhanced PI3K/AKT signaling

The observation that Lapatinib induces c-Myc is paradoxical because inhibition of a key tumor-driving HER2/AKT pathway induces a potent oncogene c-Myc, and c-Myc expression is usually inversely related to cell growth (Spencer and Groudine, 1991). Thus, we further explored whether Lapatinib or AKTi-induced c-Myc expression we observed is unique to BT474 cells or also applies to other HER2/PI3K-activated breast cancer cell lines. Many HER2+ breast cancer cell lines also harbor constitutively active mutations in PIK3CA that encodes a downstream effector of HER2 signaling, perhaps further increasing the strength of HER2 signaling.

When PI3K is hyper-activated or constitutively activated due to an active mutation, Lapatinib is no longer able to inhibit PI3K/AKT signaling, as constitutively activated PI3K can activate downstream targets (including AKTs) regardless of the active status of HER2 (Eichhorn et al., 2008). However, the inhibitor of AKTs should remain capable of inducing c-Myc expression.

Indeed, treatment of MDA-MB-361 cells, which harbor PIK3CA E545K mutation, with AKTi, but not Lapatinib (data not shown), induced expression of c-Myc (Figure 2E-F). Moreover, AKTi inhibited phosphorylation of AKT and FOXO1 precisely as Lapatinib inhibited this in BT474 cells (Figure S2D). Consistently, treatment of MCF-HER2 cells with AKTi also led to induction of c-MYC expression (Figure S2F-G).

To extend our findings to an even greater number of cell lines, we examined whether Lapatinib influences expression of c-Myc in another HER2+ (amplified) human breast cell line, UACC812 (Wang et al.). We found that Lapatinib also increased expression of c-Myc mRNA and protein levels in UACC812 cells (Figure S2H-I). Collectively, these results indicate that inhibition of the HER2/AKT pathway in multiple breast cancer cell lines induces expression of c-Myc. As Lapatinib is approved for treating breast cancer, we focused on examining the impact of Lapatinib on HER2+ cancer cells.

Lapatinib-induced c-Myc expression is critical for reducing sensitivity of breast cancer cells to Lapatinib

To determine whether Lapatinib-induced c-Myc is crucial for reducing sensitivity to Lapatinib, or is simply a biomarker responsive to the inhibition of the HER2/AKT pathway, we transduced a Doxycycline-inducible c-Myc shRNA into BT474 cells. Western blotting showed that in control cells Doxycycline led to effective knockdown of c-Myc (Figure 3A, lane 2), but did not affect cell growth (Figure 3B, column 2). Notably, Doxycycline-induced knockdown of c-Myc precluded full c-Myc induction by Lapatinib at 25 nM concentration (Figure 3A, lane 4), and significantly reduced cell number (Figure 3B, column 4). Consistently, increasing the concentration of Lapatinib to 50 nM reduced cell number compared to control (Figure 3B), and notably Doxycycline-induced c-Myc knockdown even further reduced cell growth (Figure 3B, column 6). Together, these results indicate that inducible c-Myc knockdown synergized with Lapatinib to suppress the growth of cancer cells, increasing sensitivity to Lapatinib.

Figure 3. The crucial role of Lapatinib-induced c-Myc in reducing the sensitivity of the breast cancer cells to Lapatinib.

Figure 3

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(A) The Doxycycline-inducible c-Myc shRNA lentivirus-transduced BT474 cells were treated with Doxycycline (200 nM) for 2 days, followed by treatment with various concentrations of Lapatinib for 6 days, collected 18 hr after the last treatment, prior to detection of the protein level of c-Myc by Western blotting. (B) The viable cells treated as described in (A) were counted. (C, D) BT474 cells were treated with Lapatinib (200 nM) and/or IBET (500 nM) for 4 days prior to analysis with qRT-PCR for the mRNA level (C) or with Western blotting for various proteins with the indicated antibodies (D). (E) BT474 cells were transduced with either control vector or human c-Myc-expressing lentiviruses, followed by puromycin selection and Western blot analysis for c-Myc expression. (F) Western blot analysis for the c-Myc expression in the vector control cells or c-Myc-transduced cells that were treated with Lapatinib (50 nM) and with or without IBET (500 nM). (G) The control and c-Myc-transduced cells in the presence of Lapatinib (50 nM) were treated with either DMSO or IBET (500 nM) for 4 days, followed by cell counting. Error bars indicate +/− SD. Also see Figure S3.

BRD4 is a chromatin-associating protein that can bind acetylated histones via its BET domain to upregulate gene transcription (Dey et al., 2003), and recently developed BRD4 inhibitors suppress leukemia and sarcoma partly by suppressing c-Myc expression (Dawson et al., 2011; Filippakopoulos et al., 2010). We found that IBET151 (IBET hereafter), a small molecule inhibitor of BRD4, abolished Lapatinib-induced up-regulation of c-Myc mRNA in BT474 cells (Figure 3C). Similar results were also observed in MDA-MB-361 cells after co-treatment with AKTi and IBET (Figure S3A). Consistently, Western blotting showed that IBET almost abolished Lapatinib-induced expression of c-Myc at the protein level (Figure 3D). Moreover, IBET also reduced Lapatinib-induced Bcl2 expression and increased PARP cleavage, an indicator of apoptosis, but had little effect on expression of Bim (Figure 3D). Consistently, IBET synergized with Lapatinib to suppress the breast cancer cells (Figure S3B).

As Lapatinib-mediated inhibition of HER2 represses a cascade of downstream protein kinases including PI3K and AKT, which phosphorylates and thus inhibits FOXO transcription factors by excluding their nuclear distribution (van der Horst and Burgering, 2007), we examined whether IBET has an impact on expression of FOXO1 and its cellular distribution in BT474 cells. As expected, Lapatinib increased localization of FOXO1 in the nucleus, the active fraction of FOXO1. However, IBET treatment did not change the expression or cellular distribution of FOXO1 (Fig S3C).

We ectopically expressed c-Myc in BT474 cells, and then determined whether ectopically expressed c-Myc reduces sensitivity to Lapatinib. Western blot showed that transduction of the cells with the c-Myc-expressing recombinant viruses moderately expressed c-Myc (Figure 3E). Lapatinib-induced c-Myc expression, consistently, was completely blocked by IBET in vector-transduced control cells (Figure 3F, lane 2), and correlated well with reduction of cell growth (Figure 3G, column 2). Notably, IBET failed to completely suppress c-Myc expression in the cells ectopically transduced with c-Myc (Figure 3F, lane 4 vs 2), likely because the viral promoter-driven expression of the ectopic c-Myc was independent of BRD4. In agreement, the IBET-resistant expression of ectopic c-Myc was closely correlated with the reduced sensitivity of the cells to potent suppression by a combination of Lapatinib and IBET (Figure 3G column 4 vs 2).

To control for potential off-target effects of the c-Myc shRNA, we generated a c-Myc expressing vector that contains silent mutations in the sequence that is targeted by the c-Myc shRNA. Our results showed that Doxycline modestly reduced the c-Myc protein in control cells, as expected (Figure S3D, lanes 1 and 2, bottom). Lapatinib induced c-Myc expression in vector control cells as expected (lane 3) and the expression of endogenous c-Myc was reduced by Doxycline induced c-Myc shRNA (lane 4), which correlated with Lapatinib-induced reduction of the number of the Lapatinib-treated cells (graph above lane 4).

Consistently, ectopic expression of wild-type c-Myc led to increased level of c-Myc (Figure S3D lane 5), and induction of c-Myc shRNA by Doxycycline resulted in suppression of c-Myc expression (lane 6), consistent with the reduction of the number of the Lapatinib-treated cells (graph bar above lane 6). Likewise, ectopic expression of c-Myc with the silence mutation in the shRNA target site also led to increased c-Myc protein expression (lane 7). Notably, Doxycycline-induced c-Myc shRNA failed to substantially suppress c-Myc expression, consistent with the resistance of the cells to Lapatinib-induced suppression of cell growth (graph bar above lane 8). Collectively, these results strongly suggest that the shRNA-mediated suppression of c-Myc, but not the off target effects of c-Myc shRNA, cooperates with Lapatinib to suppress the breast cancer cells.

Together, the above four lines of evidence strongly suggest the Lapatinib-induced c-Myc is crucial for reducing sensitivity of the cancer cells to Lapatinib.

A c-Myc epigenetic inhibitor synergizes with Lapatinib to suppress breast cancer cells

Consistently, IBET synergized with Lapatinib to suppress cell growth of BT474 cells, with a combination index (CI) of 0.61 (Figure 4A), where a CI < 1 indicates a synergy (Chou, 2010). IBET also synergized with AKTi to suppress growth of MDA-MB-361 (Figure 4B) and MCF-HER2 cells (Figure S4A). To assess whether the FOXO/c-Myc axis affects the activity of an AKT inhibitor in PIK3CA mutant breast cancer cells without concurrent HER2 amplification, such as T47D cells (Aksamitiene et al., 2010), we treated T47D cells with AKTi and/or IBET, and found that indeed treatment of T47D cells with AKTi alone had little effect on growth of the cancer cells up to 500 nM (Figure S4B, right panel). Notably, IBET substantially sensitized the single agent AKTi-induced suppression of the growth of T47D cells (Figure S4B, left panel). Consistently, AKTi induced c-Myc expression, which was suppressed by IBET (data not shown). These findings are consistent with the notion that Lapatinib/AKTi-induced c-Myc is crucial for reducing the cancer cell sensitivity to Lapatinib/AKTi, but suppression of c-Myc by IBET increased the sensitivity.

Figure 4. The impact of a c-Myc epigenetic inhibitor on suppressing Lapatinib-induced c-Myc expression and on synergizing with Lapatinib to suppress breast cancer cells.

Figure 4

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(A, B) BT474 (A) or MDA-MB-361 (B) breast cancer cell line was treated with IBET alone (bottom), or its combination with increasing concentrations of Lapatinib/AKTi (top), respectively, for 5 days prior to MTS assay. (C, D) BT474 (C) or MDA-MB-361 (D) cells (106) were seeded in 60 mm dishes and treated with DMSO, 100 nM lapatinib/50 nM AKTi or 500 nM IBET alone or combination of Lapatinib/AKTi with IBET for 7 days, followed by crystal violet staining. (E, F) BT474 cells transduced either with scrambled vector or BRD4 shRNA lentiviruses, followed by treatment with either DMSO or Lapatinib for 4 days, prior to measuring c-Myc expression using qRT-PCR (E) or counting viable cells (F). Error bars indicate +/− SD. Also see Figure S4.

We also treated HER2+ UACC812 cells, and found that Lapatinib-induced c-Myc expression was effectively suppressed by treatment with IBET (Figure S4C), which enhanced Lapatinib-induced suppression of the cell growth (Figure S4D). Collectively, these results strongly suggest that IBET or knockdown of IBET target synergizes with Lapatinib to suppress the breast cancer cells and that Lapatinib-induced c-Myc expression dampens sensitivity of the HER2+ breast cancer cells of to Lapatinib.

Furthermore, colony formation assay showed that IBET also synergized with Lapatinib to suppress BT474 cells (Figure 4C), and with AKTi to suppress MBA MB 361 cells (Figure 4D). Altogether, these results indicate that IBET-mediated suppression of c-Myc, at least partly, contributes to increasing sensitivity, or reducing resistance, to Lapatinib. We also found that knockdown of BRD4, a target of IBET, also abolished Lapatinib-induced expression of c-Myc (Figure 4E) and also reduced cell growth. Notably BRD4 knockdown further reduced the Lapatinib-induced suppression of cell growth (Figure 4F).

FOXOs mediate Lapatinib-induced expression of c-Myc

While we have shown that Lapatinib or AKTi-induced c-Myc is crucial for reducing Lapatinib sensitivity, little is known as to how Lapatinib induces c-Myc expression. We noticed that Lapatinib not only markedly increased expression of c-Myc, but also dramatically increased expression of total FOXO1 (Figure S2C) and reduced FOXO1 phosphorylation (Figure 2B). This observation of close association of FOXO1 and c-Myc regulation prompted us to investigate whether FOXOs affects c-Myc expression. However, a link between FOXO activation and c-Myc expression is paradoxical and unexpected, as normally FOXOs are tumor-suppressing factors while c-Myc is a bona fide proto-oncogene, and in fact FOXOs and c-Myc mutually antagonize each other in various settings (Bouchard et al., 2007; Peck et al., 2013).

Notably, when we treated BT474 cells with a small molecule FOXO1 inhibitor that binds to unphosphorylated FOXO1 and decreases FOXO1 transactivation (Nagashima et al., 2010), Lapatinib-induced expression of c-Myc and Bcl2 was markedly reduced at both the mRNA and protein levels (Figure 5A and Figure S5A). We further found that FOXO1 knockdown substantially reduced Lapatinib-induced c-Myc expression (Figure 5B). Knockdown of both FOXO1 and 3 moderately reduced cell growth, but further reduced Lapatinib-mediated suppression of the cell growth (Figure 5C). Consistently, FOXO1/3 knockdown also potently blocked Lapatinib-induced c-Myc expression at both the mRNA and protein level (Figure 5D-E). Similarly, FOXO1/3 knockdown also blocked Lapatinib-induced expression of Bcl2 (Figure S5B and Figure 5E).

Figure 5. The role of FOXOs and MLL2 in mediating Lapatinib-induced expression of c-Myc.

Figure 5

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(A) BT474 cells were treated with FOXO1 inhibitor, AS1842856, and/or Lapatinib for 4 days, and subjected to analysis for c-Myc mRNA by qRT-PCR or the protein level with Western blot. (B) The cells were transduced with control or FOXO1 shRNAs, and the resulting stable cells were subjected to analysis of mRNAs for FOXO1 or the c-Myc. (C-E) The control or FOXO1/3 KD cells were treated with DMSO or Lapatinib for 4 days, followed by analyzing the number of viable cells (C), c-Myc expression (D), or protein levels of c-Myc, Bcl2, and FOXOs (E). (F, G) Control or MLL2 shRNA-transduced BT474 cells were treated with either DMSO or Lapatinib, followed by cell counting (F) or qRT-PCR for c-Myc expression (G). Error bars indicate +/− SD. Also see Figure S5.

To extend our findings to additional HER2 amplified or PIK3CA mutated cell lines, we knocked down expression of FOXO1/3 in MDA-MB-361 cells, and found that the FOXO knockdown decreased ATKi-induced c-Myc expression (Figure S5C). Consistently, knockdown of FOXOs in HER2+ UACC812 cells, while modestly, decreased Lapatinib-induced expression of c-Myc (Figure S5D). Moreover, the FOXO inhibitor (FOXOi) also reduced Lapatinib-induced c-Myc expression (Figure S5E). Together, these results demonstrate that FOXO1/3 are required for Lapatinib-induced c-Myc expression and their knockdown sensitizes the cancer cells to Lapatinib-induced suppression of cell growth.

MLL2 and its partners are crucial for reducing sensitivity to Lapatinib

We showed that MLL2 was crucial for expression of c-Myc, but not Bclxl, and we thus determined whether MLL2 is also crucial for Lapatinib-induced c-Myc expression. We found that MLL2 knockdown in BT474 cells reduced cell growth, and Lapatinib further reduced the growth of MLL2 knockdown cells (Figure 5F). Consistently, MLL2 knockdown also markedly suppressed Lapatinib-induced upregulation of the c-Myc mRNA (Figure 5G). It is currently unclear what precise proportion of the effects of MLL2 knockdown on cell viability is due to loss of c-Myc expression. Moreover, knockdown of GCN5, a histone acetyltransferase, also suppressed Lapatinib-induced expression of c-Myc (Figure S5F). These findings suggest that epigenetic regulator MLL2, its partners, and GCN5 play a crucial role in Lapatinib-induced expression of c-Myc and reduction of sensitivity to Lapatinib treatment.

Lapatinib induces direct binding of FOXOs to the c-Myc promoter to promote c-Myc expression

We explored whether FOXOs directly bind the c-Myc locus. Notably, a survey of DNA sequence in the c-Myc locus revealed a consensus “TGTTTAC” sequence of the FOXO binding element (FBE) in the c-Myc promoter that is conserved across multiple species (Figure 6A). Chromatin immunoprecipitation (ChIP) assay indicated that Lapatinib induced binding of FOXO1/3 to the c-Myc promoter in BT474 cells (Figure 6B). To further determine whether the FOXO binding site is crucial for FOXO-mediated gene transcription, we constructed a luciferase reporter driven by either the wild-type FBE from the c-Myc promoter or its mutant form, and transfected them into BT474 cells, followed by Lapatinib treatment and detection of the mRNA of the reporter gene. We found that Lapatinib increased expression of the reporter driven by the wild-type FBE four fold, but not by the mutant FBE (Figure 6C), indicating that the FBE in the c-Myc promoter is required for FOXO-mediated expression of c-Myc.

Figure 6. The role of Lapatinib in regulating binding of FOXOs, recruitment of the histone modifiers, and histone modifications at the c-Myc locus.

Figure 6

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(A) A conserved FOXO binding element (FBE) in the promoter of the c-Myc gene in various species. (B) ChIP analysis for binding of FOXO1 and FOXO3 to the c-Myc promoter in BT474 cells upon Lapatinib treatment for 3 days, followed by 18 hr fresh treatment. (C) BT474 cells transfected with a luciferase reporter driven by either the wild-type FBE or the mutant form (Top panel), followed by treatment with Lapatinib for 18 hr prior to qRT-PCR analysis for the reporter expression. (D-F) The control and Lapatinib treated cells were subjected to ChIP assay for detecting binding of MLL2 (D) and GCN5 (E) as well as H3K4m3 and histone acetylation (F) at the c-Myc locus. (G) BT474 cells were treated with FOXOi and/or Lapatinib for 4 days, followed by ChIP assay for detecting MLL2 binding (left) or H3K4m3 and histone acetylation (right) at the c-Myc locus. (H) The cells were treated with Lapatinib and/or IBET for 4 days, collected 18 hr after last treatment followed by ChIP assay to detect BRD4 binding to the c-Myc locus. Error bars indicate +/− SD. Also see Figure S6.

Importantly, Lapatinib induced the recruitment of MLL2 to the c-Myc promoter (Figure 6D) and increased detection of GCN5 (Figure 6E) and histone H3 lysine 4 trimethylation (H3K4m3) and histone H3 acetylation (Figure 6F), both histone markers for active gene transcription, at the c-Myc locus.

ChIP assay showed that FOXOi did not affect FOXO binding to the c-Myc promoter (data not shown), but reduced Lapatinib-induced recruitment of MLL2 and RNA polymerase II, H3K4m3, and histone acetylation at the c-Myc locus (Figure 6G and Figure S6A). Similarly, FOXOi also reduced the recruitment of MLL2 to the Bcl2 locus (Figure S6B) and the level of H3K4m3 mark and histone acetylation at the locus, while not affecting the level of total histone H3 (Figure S6C). We also found BRD4, at the c-Myc promoter in cells treated with Lapatinib, but IBET potently inhibited binding of BRD4 to the c-Myc locus (Figure 6H), while not affecting FOXO1 translocation to the nucleus (Figure S3C, lane 4). Consistently, binding of FOXO1/3 or MLL2 to the c-Myc promoter was also induced by Lapatinib in HER2+ UACC812 cells as shown by ChIP assay (Figure S6D). Collectively, these results indicate that Lapatinib induces FOXO binding to the c-Myc promoter, enhances the recruitment of epigenetic regulators including MLL2 to the c-Myc promoter, and increases histone acetylation and recruitment of BRD4 at the c-Myc locus. This coordinated action may lead to increased expression of target genes such as c-Myc, and at least partly results in reduced sensitivity to Lapatinib. Inhibition of BRD4 binding reduces transcription of c-Myc but increases the sensitivity to Lapatinib in the breast cancer cells, and synergizes with Lapatinib to suppress the growth of breast cancer cells.

Lapatinib and IBET synergistically suppress HER2+ cancer in xenograft models

To determine whether a combination of Lapatinib and IBET synergizes to suppress the HER2+ breast cancer in xenograft models, we first induced tumors in immunodeficient mice with transplantation of BT474 cells. When the tumors were formed visibly, we started to treat the mice with control vehicle, Lapatinib, IBET, or a combination of Lapatinib and IBET. At the end of the observation, the tumors were collected, and used for making RNA or for immunohistochemistry analysis.

We found that treatment of the mice with either Lapatinib or IBET alone moderately slowed the tumor growth (Figure 7A). Notably, a combination of Lapatinib and IBET significantly reduced growth of the tumor (Figure 7A). The body weight of the mice in 4 groups of the mice was not substantially changed (data not shown). Consistently, Lapatinib also induced expression of c-Myc, but treatment with IBET blocked c-Myc and Bcl2 induction in the collected tumor samples, as shown by qRT-PCR (Figure 7B). While the Lapatinib and IBET combination did not show marked reduction in the tumors in size (Figure 7A), H&E staining of the tumor tissue showed that the number of cells was substantially reduced in the tumor treated with the combination, filled with fibrosis-like tissue (Figure 7C). Consistently, immunofluorescent staining showed that c-Myc was induced in the tumor cells treated with Lapatinib, but the induction was quenched by IBET treatment (Figure 7D).

Figure 7. The impact of a combination of Lapatinib and IBET on suppressing HER2+ cancer in xenograft models.

Figure 7

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(A) Nude mice were transplanted with 10 × 106 BT474 cells, 16 days after transplantation, when the tumors were established, the mice with tumor size > 150 mm3 (n=6 per group) were treated with control saline, or Lapatinib (25 mg/kg on alternate days by oral gavage) and/or IBET (15 mg/kg IP injection daily), for the indicated length of days. The size of the tumors was measured with a vernier caliber every 3 days. Error bars indicate +/− SEM. (B) Tumors from 4 mice from each group were collected 16 hr after last treatment, at the end of study and used to isolate RNAs, which were used to detect the mRNA levels of c-Myc or Bcl2. Error bars indicate +/− SD. (C) Representative H&E staining of the transplanted tumors from each of the 4 groups. The measurement bar denotes 50 μM. (D) Immunofluorescent staining for c-Myc from tumors treated with the indicated conditions. The bar denotes 200 μM. Also see Figure S7.

We also performed a similar tumor xenograft experiment using another HER2-amplified cell line, UACC812. Consistently, we found that treatment of the mice bearing UACC812 cell-derived tumors with either Lapatinib or IBET alone only moderately suppressed the tumor growth (Figure S7A). Notably, a combination of Lapatinib and IBET led to significant suppression of the tumor burden (Figure S7A) Lapatinib upregulated expression of c-Myc, as shown by immunohistochemistry staining, but IBET treatment substantially reduced Lapatinib induced c-Myc expression (Figure S7B). Together, these in vivo studies strongly suggest that targeting the MLL2/FOXO/BRD4/c-Myc axis may represent a great avenue to increase sensitivity and reduce resistance to inhibitors of the HER2/AKT pathway, leading to marked improvement of therapy.

DISCUSSION

We took an unbiased shRNA screening approach and identified multiple components from the MLL2 complex that are crucial for the maintenance of HER2+ BT474 breast cancer cells. By investigating the potential role of the MLL2 complex and associating factors in response of the breast cancer cells to inhibitors of the HER2/AKT pathway, we uncovered an important link between the epigenetic MLL2 complex/FOXO/c-Myc axis and reducing sensitivity of HER2+ breast cancer cells to the HER2 inhibitor, Lapatinib. These findings are crucial and significant in the following aspects: First, as the HER2/PI3K/AKT pathway is often upregulated or activated in a broad spectrum of cancers (Courtney et al., 2010), and targeting the kinases of this pathway is promising yet thus far has only yielded limited success due to quick development of reduced sensitivity (Chandarlapaty et al., 2011; Muranen et al., 2011), our findings of an intrinsic and epigenetic pathway that partially accounts for the reduced sensitivity unravels multiple epigenetic components that could be targeted to improve therapy.

Second, as FOXOs and c-Myc are normally considered tumor suppressor and oncogene respectively (Bouchard et al., 2007; Peck et al., 2013), our findings that FOXOs positively regulate c-Myc to reduce sensitivity of the cancer cells to Lapatinib or AKTi provide insights into how tumor suppressing FOXOs act in concert with c-Myc to reduce the sensitivity to Lapatinib in the breast cancer cells. These findings suggest means to sensitize the HER2+ or PI3K/AKT pathway-activated breast cancer cells to the inhibitors to improve the therapy.

Third, mechanistically, we found that the MLL2/FOXO axis regulates transcription of c-Myc mRNA in concert with a cascade of other chromatin modifying or associating proteins such as GCN5 and BRD4. Finally, as a proof of principle based on these mechanistic findings (Figure 8), we found that an epigenetic inhibitor against BRD4, IBET, synergizes with Lapatinib at lower concentrations to suppress HER2+ cancer in vitro and in vivo. It is conceivable that multiple components in this pathway can also be similarly targeted in combination with the HER2/PI3K/AKT inhibitors to cause synthetic lethality in other types of cancer cells and improve therapy.

Figure 8.

Figure 8

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A model summarizing the epigenetically regulated MLL2/FOXO/BRD4/c-Myc- axis in reducing the sensitivity of the HER2+ breast cancer cells to Lapatinib, and the synergy of Lapatinib and IBET in suppressing HER2+ breast cancer.

We uncovered that multiple epigenetic regulators, including components from the MLL2 epigenetic complex such as MLL2, WDR5, RBBP5 and GCN5 are crucial for growth of BT474 cells. These proteins upregulate expression of multiple pro-proliferative/survival targets, including c-Myc and Bcl2. In contrast, other methyltransferases such as MLL3 and MLL4 did not affect growth of the breast cancer cells, indicating a specific role for MLL2 in regulating these target genes in breast cancer cells. Although loss-of-function mutations in MLL2 lead to inherited Kabuki syndrome and follicular lymphoma (Makrythanasis et al.; Morin et al., 2011), a recent report suggests that MLL2 plays an important role in maintenance of HER2+ colon cancer cells via upregulating ITGB4 and NRG1 (Guo et al., 2012). Thus the function of MLL2 and its related proteins in regulating cancer cells may be cell-type or signaling pathway-specific. It is also possible that other epigenetic regulators, while not the components of the MLL2 complex (such as GCN5, BPTF, and BRDs), are also involved in the process because they are also crucial for growth of the HER+ cancer cells. These findings pave the way to investigate whether this epigenetic pathway is also crucial for regulating the cancer cell sensitivity to other inhibitors of the HER2/PI3K/AKT pathway.

While the HER2/PI3K/AKT pathway is often upregulated or activated in multiple types of cancers (Courtney et al., 2010), targeted inhibition of this pathway has only met limited success due to reduced sensitivity via various mechanisms including up-regulation of RTKs (Chandarlapaty et al., 2011; Garrett et al., 2011; Muranen et al., 2011). Our results suggest that HER2/AKT inhibitor-induced FOXO/c-Myc is an important axis that reduces sensitivity to the inhibitors, as these inhibitors induced c-Myc expression in multiple HER2+ or PIK3CA-mutated human breast cancer cell lines, and down-regulating c-Myc expression synergizes with Lapatinib to suppress HER2+ breast cancer cells.

Our data also suggest that temporally in the early phase of treatment Lapatinib suppressed cell growth and survival, likely partly via upregulating expression of Bim and CDKI like p27kip by Lapatinib-activated FOXOs (Greer and Brunet, 2005). In the late phase of treatment after 3-6 days, on daily treatment with Lapatinib, expression of c-Myc is further upregulated by marked upregulation of FOXOs in concert with other epigenetic regulators including the MLL2 complex, thereby reducing sensitivity to Lapatinib. This may also partly explain that c-Myc is important for tumor dormancy after treatment (Bellovin et al., 2013), as Lapatinib may lead to cell killing in early treatment but its chronic effect on c-Myc induction may facilitate development of tumor dormancy. Other pathways including Estrogen receptor signaling and Lapatinib-induced RTKs are also likely involved in regulating or crosstalk with the FOXO/c-Myc axis in response to HER2 inhibition. However, we cannot rule out the possibility that the synergistic effect of Lapatinib and IBET also involves additional factors beyond c-Myc. The Lapatinib-induced c-Myc expression reduces the drug sensitivity likely partly via multiple pathways including regulating ribosomal biogenesis, as well as reprogramming the cancer metabolic pathways and promoting survival (van Riggelen et al., 2010).

It is noteworthy that inhibition of the HER2/AKT pathway inevitably leads to activation of FOXOs, which are normally tumor-suppressing factors (Zhang et al., 2011), by reducing FOXO phosphorylation. FOXOs antagonize c-Myc in renal cell carcinoma RCC4 cells, possibly by increasing the expression of miR-145 (Gan et al., 2010). Thus, our findings define an intrinsic adaptive pathway via FOXO/c-Myc to reduce drug sensitivity.

Our results show that the normally mutually antagonistic FOXOs and c-Myc cooperate to reduce sensitivity to Lapatinib in breast cancer cells with enhanced HER2/PI3K signaling. While constitutively high expression or amplification of c-Myc in a subset of breast cancer cells results in resistance to PI3K inhibitor (Ilic et al., 2011), our findings indicate that in the breast cancer cells with enhanced HER2/PI3K signaling the basal level of c-Myc is not crucial for maintenance of the cells. Rather, Lapatinib-induced c-Myc expression mediated by MLL2/FOXO is responsible for reducing sensitivity to Lapatinib. These findings raise an attractive possibility that inhibiting MLL2/FOXOs/c-Myc axis synergizes with the HER2/PI3K/AKT inhibitors to suppress HER2+ breast cancer and likely other PI3K/AKT-activated cancers as well. Conceivably, preemptively targeting this common and intrinsic pathway that reduces the drug sensitivity may be a good strategy to improve the therapeutic efficacy of the HER2/AKT/PI3K inhibitors.

Our findings suggest the following model: First, Lapatinib or PI3K/AKT inhibitor suppresses the kinases, leading to activation of FOXOs, which in turn translocate into the nucleus and recruits MLL2 and GCN5 to their target gene, c-Myc, to add active histone marks, including H3K4m3 and histone acetylation. The modified histones can further recruit proteins such as BPTF, an H3K4m3 binding protein, and BRD4, an acetylated histone-binding protein, to the target genes, leading to increased gene transcription (Figure 8). MLL and GCN5 complexes can both be recruited to an Myc gene to epigenetically upregulate its expression (Choi and Boss, 2012). It is likely that FOXO and/or FOXO-mediated MLL2 is also conducive to recruitment of GCN5 and histone acetylation at the locus. While extensive work was carried out to unravel the epigenetic pathway in regulating the sensitivity of HER2+ breast cancer cells to the HER2 and AKT inhibitors, the detailed mechanisms governing their crosstalk of the pathway's components remain to be further investigated. Nevertheless, these players are crucial for reducing the sensitivity of the cancer cells to Lapatinib as knockdown of FOXO1/3, MLL2 and BRD4 potentiated Lapatinib-induced suppression of the cancer cells.

IBET was not very effective as a single agent in inhibiting HER2+ breast cancer cells with the dose used in our study. Rather, a combination of Lapatinib with IBET synergistically suppressed HER2+ breast cancer cells in cell culture more effectively at lower concentrations of Lapatinib/AKTi and in a mouse model. This combination blocks Lapatinib-induced upregulation of c-Myc, and it is conceivable that IBET can also inhibit expression of other genes besides c-Myc that are induced by Lapatinib such as HER3. As such, it is possible that inhibitors of epigenetic regulators involved in the MLL2/FOXO/c-Myc axis, such as MLL2, WDR5, BPTF, and GCN5 could also be targeted to improve HER2/PI3K/AKT inhibitor-mediated therapy. While a combination of Lapatinib and IBET also suppressed growth of UACC812 cell-derived tumors more effectively, the combination did not eliminate the tumor. It is possible that the cancer cells were influenced by additional microenvironmental factors during the treatment. Alternatively, it is also possible that better dosing and timing is needed to further increase the efficacy. These results also indicate that multiple components in the epigenetic MLL2/FOXO axis could be preemptively or simultaneously targeted with the HER2/PI3K/AKT inhibitors to cause synthetic lethality of the HER2+ breast cancers. Other types of tumors with the constitutively active RTK/PI3K/AKT pathway may also be susceptible to this strategy.

EXPERIMENTAL PROCEDURES

Cell lines, cell culture, drug treatment, and plasmids

UACC812 cell line, which contains HER2 amplification, MDA-MB-361, and T47D cell lines, which harbor a PIK3CA active mutation (Aksamitiene et al., 2010), were purchased from ATCC. BT-474 cell line was a gift from Dr. Lewis Chodosh, MCF-HER2 and MCF-Neo cell lines were gifts from Dr. Mien-Chi Hung. To generate a cell line with conditional expression of c-Myc shRNA, shRNA oligonucleotides for c-Myc was cloned into pLKO-tet-on vector from Addgene. BT474 cells were transduced with either scramble or shRNA targeted against c-Myc mRNA. Cells were selected with puromycin. Wild-type c-Myc was cloned in MSCV-neo vector from Addgene. To obtain c-Myc impervious to shRNA knockdown site directed mutagenesis of this plasmid was performed to change 5’-CAGCAAC to 5’-ATCGAAT using a kit from Stratagene. All cell lines were cultured in DMEM with 10% FBS and 1% Penicillin/ Streptomycin and maintained at 37° C and 5% CO2.

Immunoblotting

Lysates from cells in culture were prepared by washing twice in cold PBS followed by lysis with either SDS-lysis buffer (20 mM Tris-HCl (pH8), 100 mM NaCl, 1% SDS) or RIPA-lysis buffer supplemented with protease and phosphatase inhibitors. For lysis in SDS, lysates were boiled for 5 minutes followed by brief sonication. Lysates were cleared by centrifugation at 14,000×g (10 min) and the supernatant was collected. Protein concentration of each sample was determined using the BCA kit (Pierce) per manufacturer's instructions. Proteins (25 or 50 μg) was loaded onto 4- 10% SDS-PAGE minigels for immunoblotting.

qRT-PCR

Total RNA was extracted from cultured cells using Trizol as previously reported (Thiel et al., 2010). RNA (1 μg) was used to make cDNA. Real time PCR was performed using 7500 fast real time PCR System. SDS software was used for data analysis. Sequence of the probes used can be found in supplemental information.

Breast cancer xenograft in mice

All laboratory mice were maintained on a 12 hr light-dark cycle in the animal facility at the University of Pennsylvania. All experiments on mice in our research protocol were approved by Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania and were performed in accordance with relevant institutional and national guidelines and regulations. Six to 8 week old nu/nu athymic female mice were obtained from NCI-Frederick Cancer Center. Mice were implanted with 0.72 mg sustained release estradiol pellets (Innovative Research of America) three days prior to implantation of cells. 10 × 106 BT474 or UACC812 cells with matrigel (1:1) were injected into right flank of the mouse. Mice with tumor size ~100 mm3 were randomized into four groups and treated with vehicle, Lapatinib, IBET or Lapatinib + IBET with the indicated doses. Tumor dimensions were measured with vernier calipers and tumor volume was calculated as ½ larger diameter × (smaller diameter) 2.

Statistical analysis

Microsoft Excel and GraphPad Prism software were used for statistical analysis. Student's t-test and Annova were used to determine the significance of the results.

Supplementary Material

supplemental materials

SIGNIFICANCE.

While the HER2/PI3K/AKT pathway is frequently mutated in a wide range of cancers, thus driving tumorigenesis, inhibitors of this pathway such as Lapatinib yield only limited success, as the cancer cells often adapt quickly. We found that the MLL2/FOXO/c-Myc axis was activated by Lapatinib, reducing sensitivity to the drug. These findings not only uncover an intrinsic adaptation pathway comprising the normally mutually antagonizing players, FOXOs and c-Myc, that are regulated by multiple epigenetic regulators, but also highlight several key components in the pathway for future targeting to improve therapy. As a proof of principle, a BRD4 inhibitor and Lapatinib synergistically suppressed HER2+ breast cancer cells in vitro and in vivo, at least partly, by targeting the MLL2/FOXO/c-Myc axis.

Acknowledgments

We thank Drs. Peter Zhou and Yong Wan for critically reading the manuscript, Brian Bakke and Haoren Wang for technical assistance, and Ashley Banks for editing the manuscript. This work was supported in part by grants from the NIH (R01-DK085121) (1-R01-CA-178856 and R01 DK097555), Caring for Carcinoid Foundation-AACR Grant Care for Carcinoid Foundation (11-60-33), a pilot grant from ITMAT of the University of Pennsylvania, as well as a sarcoma pilot grant from University of Pennsylvania and Pennsylvania Breast Cancer Refunds for Research (PABCC). This work was also supported in part by the NIH/NIDDK Center for Molecular Studies in Digestive and Liver Diseases (P30DK050306) and its core facilities (Molecular Biology/Gene Expression Core). We thank Austin Thiel for help in assembling the epigenetic shRNA library and John Tobias for statistical analysis.

Footnotes

Author contributions

S. M. and X.H. designed the experiments and wrote the manuscript. S.M. performed majority of the experiments. P.S. performed some RT-PCR and Western blots. S.G. performed some mouse xenograft experiments. B.G. performed immunofluorescence assay. B.K. helped revising the manuscript. J.L. validated shRNA screen targets. A.B.M. performed flow cytometry experiments. X.K. performed some RT-PCR and Western blots. L.W. performed Immunohistochemistry staining. G.J. designed and supervised some mouse xenograft experiments. C.D. helped design some experiments.

Additional EXPERIMENTAL PROCUDURES can be found in the supplemental materials.

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