MicroRNA-101 Suppresses Tumor Cell Proliferation by Acting as an Endogenous Proteasome Inhibitor via Targeting the Proteasome Assembly Factor POMP.

Article

MicroRNA-101 Suppresses Tumor Cell Proliferation by Acting as an Endogenous Proteasome Inhibitor via Targeting the Proteasome Assembly Factor POMP Graphical Abstract

Authors Xin Zhang, Ramona Schulz, Shelley Edmunds, ..., Arnold J. Levine, Ute M. Moll, Matthias Dobbelstein

Correspondence [email protected]

In Brief The proteasome represents a cancer drug target, and its inhibition results in the accumulation of tumor-suppressive proteins. Zhang et al. identify a microRNA that targets the proteasome maturation factor POMP and interferes with proteasome assembly. It thus serves as an endogenous proteasome antagonist and a suppressor of cancer cell survival.

Highlights d

miR-101 functions as an endogenous proteasome inhibitor by targeting POMP

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Targeting POMP is essential for cell growth suppression by miR-101

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High miR-101 levels have good outcomes for ERa-positive breast cancer patients

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Targeting POMP inhibits tumor progression and overcomes resistance to bortezomib

Zhang et al., 2015, Molecular Cell 59, 1–15 July 16, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2015.05.036

Accession Numbers GSE69150

Please cite this article in press as: Zhang et al., MicroRNA-101 Suppresses Tumor Cell Proliferation by Acting as an Endogenous Proteasome Inhibitor via Targeting the Proteasome Assembly Factor POMP, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.05.036

Molecular Cell

Article MicroRNA-101 Suppresses Tumor Cell Proliferation by Acting as an Endogenous Proteasome Inhibitor via Targeting the Proteasome Assembly Factor POMP Xin Zhang,1 Ramona Schulz,1 Shelley Edmunds,1 Elke Kru¨ger,2 Elke Markert,3 Jochen Gaedcke,4 Estelle Cormet-Boyaka,5 Michael Ghadimi,4 Tim Beissbarth,6 Arnold J. Levine,7 Ute M. Moll,1,8 and Matthias Dobbelstein1,* 1Institute

of Molecular Oncology, University Medical Center Go¨ttingen, 37077 Go¨ttingen, Germany fu¨r Biochemie, Charite´-Universita¨tsmedizin Berlin, 10117 Berlin, Germany 3Cancer Research UK Beatson Institute, Glasgow, G61 1BD Scotland, UK 4Department of General, Visceral and Pediatric Surgery, University Medical Center Go ¨ ttingen, 37075 Go¨ttingen, Germany 5Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA 6Department of Medical Statistics, University Medical Center Go ¨ ttingen, 37075 Go¨ttingen, Germany 7Institute for Advanced Study, Einstein Drive, Princeton, NJ 08540, USA 8Department of Pathology, Stony Brook University, Stony Brook, NY 11794, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.05.036 2Institut

SUMMARY

Proteasome inhibition represents a promising strategy of cancer pharmacotherapy, but resistant tumor cells often emerge. Here we show that the microRNA-101 (miR-101) targets the proteasome maturation protein POMP, leading to impaired proteasome assembly and activity, and resulting in accumulation of p53 and cyclin-dependent kinase inhibitors, cell cycle arrest, and apoptosis. miR-101-resistant POMP restores proper turnover of proteasome substrates and re-enables tumor cell growth. In ERa-positive breast cancers, miR-101 and POMP levels are inversely correlated, and high miR-101 expression or low POMP expression associates with prolonged survival. Mechanistically, miR-101 expression or POMP knockdown attenuated estrogen-driven transcription. Finally, suppressing POMP is sufficient to overcome tumor cell resistance to the proteasome inhibitor bortezomib. Taken together, proteasome activity can not only be manipulated through drugs, but is also subject to endogenous regulation through miR-101, which targets proteasome biogenesis to control overall protein turnover and tumor cell proliferation. INTRODUCTION The ubiquitin-proteasome system (UPS) utilizes a highly diversified network of ubiquitin ligases to determine turnover rates of individual proteins. Protein stability is also determined by the overall activity of the proteasome. The 26S proteasome is a multi-subunit protein complex, which consists of a 20S catalytic core unit covered by 19S regulatory particles on both sides

(Murata et al., 2009). The 20S core unit has a cylindrical structure made of four stacked rings. The two inner rings comprise seven b subunits each, three of which have enzymatic activities, characterized as caspase-like, trypsin-like, and chymotrypsin-like (CT-L), respectively. The two outer rings comprise seven a subunits each, serving as an entry gate into the catalytic chamber. Proteasome biogenesis is a highly coordinated multistep event involving the synthesis of all subunits, proteasome assembly, and maturation. Proteasome assembly is not autonomous, but requires chaperones (Murata et al., 2009). In yeast, Ump1 was identified as an assembly factor for the 20S proteasome (Ramos et al., 1998). Ump1 is transiently incorporated into the 20S proteasome precursor, where it mediates the processing of b-subunits and dimerization of half-proteasomes. Subsequently, Ump1 is degraded within the newly synthesized 20S proteasome. The human homolog of Ump1 was named proteasome maturation protein (POMP), hUMP1, or proteassemblin (Burri et al., 2000; Griffin et al., 2000; Witt et al., 2000). Human POMP is required for the recruitment of b-subunits to the a-ring, together forming the half-structure of the 20S proteasome (also called ‘‘16S proteasome precursor’’) (Heink et al., 2005; Fricke et al., 2007; Hirano et al., 2008). In general, cancer cells are more susceptible to proteasome inhibition than normal cells, providing a basis for therapeutic exploitation (Masdehors et al., 1999; Hideshima et al., 2001). Proteasome inhibitors belong to an emerging class of anticancer drugs that target a tumor-supportive molecular machinery (Dobbelstein and Moll, 2014). Bortezomib is a proteasome inhibitor approved by the FDA for the treatment of multiple myeloma (MM) and mantle cell lymphoma (Richardson et al., 2005; Fisher et al., 2006). It can reversibly bind to the b5 subunit in the 20S proteasome and inhibit CT-L activities (Ruschak et al., 2011). However, despite the success of bortezomib in the treatment of selected hematological malignancies, its efficacy against solid tumors so far is less encouraging (Hainsworth et al., 2007; Irvin Molecular Cell 59, 1–15, July 16, 2015 ª2015 Elsevier Inc. 1


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Figure 1. miR-101 Acts as an Endogenous Proteasome Inhibitor (A) Immunoblot analysis of p53 in U2OS, HCT116, and MCF7 cells transfected with scrambled miRNA (scr) or miR-101. b-actin serves as loading control. The intensities of the bands were quantitated by ImageJ, and the relevant results are displayed beneath the bands. (B) miR-101-mediated accumulation of modified p53 as a function of Mdm2. U2OS cells were transfected with control siRNA (si ctrl) or Mdm2 siRNA (si Mdm2). At 24 hr post-transfection, cells were further transfected with scr or miR-101 and incubated for another 48 hr. (C) Detection of HA-ubiquitinated p53 upon miR-101 transfection. p53 was immunoprecipitated using anti-HA antibody in scr- or miR-101-transfected U2OS cells that stably express HA-tagged ubiquitin and was analyzed by immunoblot. (legend continued on next page)

2 Molecular Cell 59, 1–15, July 16, 2015 ª2015 Elsevier Inc.


et al., 2010). Furthermore, besides pharmacological proteasome inhibitors (Buac et al., 2013), little is known regarding whether and how endogenous regulators can modulate the overall activity of the proteasome to suppress cell growth. MicroRNAs (miRNAs) represent master regulators of gene expression. Through base pairing with the complementary 30 untranslated regions (30 UTRs) of their target mRNA molecules, miRNAs mediate mRNA degradation and/or suppress translation. miRNAs are involved in a broad panel of diseases including cancer (Croce, 2009). miR-101 functions as a tumor suppressor and is downregulated in a variety of malignancies (Varambally et al., 2008; Su et al., 2009; Kottakis et al., 2011). In contrast, miR-101 restoration prevents cancer cell proliferation. This potent anti-tumor activity was previously ascribed to the ability of miR-101 to suppress the synthesis of EZH2 (Varambally et al., 2008), the human homolog of the Drosophila enhancer of zeste gene product. EZH2 is a major component of the polycomb repressor complex 2 that mediates the trimethylation of histone H3 at lysine 27. This histone modification supposedly represses the transcription of differentiation-associated genes, allowing the cells to maintain a stem cell-like phenotype, along with tumorigenic properties. In this view, miR-101 would counteract tumor cell stemness and thereby antagonize tumor progression by targeting EZH2. In this study we present unexpected evidence that miR-101 is a negative regulator of proteasome biogenesis by targeting POMP. Our work reveals the existence of an endogenous proteasome inhibitor, capable of suppressing tumor cell growth and acting like pharmacological proteasome inhibitors. RESULTS miR-101 Acts as an Endogenous Proteasome Inhibitor Since miR-101 is known to exert potent tumor-suppressive functions, we asked whether miR-101 cross-talks with the pathways activating the tumor suppressor p53. To this end, we determined p53 protein levels after introducing synthetic miR-101 into different tumor cell lines harboring wild-type p53 (U2OS, MCF7, and HCT116). Indeed, upon transfection of miR-101, p53 accumulated in all cell lines (Figure 1A). Interestingly, miR101 induced protein ‘‘ladders’’ resembling ubiquitinated p53. Moreover, removing the p53-specific ubiquitin ligase Mdm2 led to a loss of the p53 ladder (Figure 1B). This suggested that miR-101 interferes with the degradation of ubiquitinated p53 by the proteasome. To further test if this modification consists of ubiquitin, we established a U2OS cell line that stably expresses HA-tagged ubiquitin. After transfection with miR-101 and immunoprecipitation using an anti-HA antibody, we found enriched HA-ubiquitinated p53 (Figure 1C), strongly suggesting that miR-101 causes the accumulation of ubiquitinated p53. Besides p53, we also observed miR-101-dependent accumulation

of other proteins that normally undergo rapid UPS turnover, including CDKN1A/p21, Mdm2, and CDKN1B/p27 (Figure 1D) (Pagano et al., 1995; Blagosklonny et al., 1996; Chang et al., 1998). Their protein accumulation was not accompanied by any significant increase in their corresponding mRNA levels (Figure 1E). Instead, cycloheximide chase revealed that miR-101 increased the stability of these proteins (p53, p21, and Mdm2; Figure 1F). Thus, the stabilization of multiple proteins suggests that miR-101 impairs proteasome activity, leading to accumulation of its substrates. Indeed, poly-ubiquitinated proteins strongly accumulated upon ectopic miR-101 expression (Figure 1G). Taken together, we propose that miR-101 represents an endogenous proteasome inhibitor. miR-101 Targets the Proteasome Maturation Protein POMP Array hybridization was performed to determine changes in mRNA levels upon ectopic expression of miR-101. Numerous genes were found downregulated by miR-101 (Tables S1 and S2). Although the ubiquitin/proteasome system was not among the major terms found by GO analysis (Table S3), we identified one gene targeted by miR-101 as a candidate to explain the accumulation of ubiquitinated proteins. This gene encodes the proteasome maturation protein POMP (Figure 2A), which is essential for proper proteasome assembly (Heink et al., 2005). As shown in Figure S1, POMP knockdown was sufficient to stabilize p53, p21, Mdm2, and p27 and to accumulate poly-ubiquitinated proteins. Thus, POMP knockdown phenocopied miR101 expression. As shown in Figures 2B and 2C, miR-101 downregulated POMP in the cell lines U2OS, HeLa, MCF7, and HCT116, too. To assess the impact of endogenous miR-101 on POMP levels, we employed HepG2 cells treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), which lead to enhanced miR-101 expression in a previous (Chiang et al., 2010) study and in our study (Figure S2A). We found that endogenous POMP (as well as EZH2) levels were decreased by TPA (Figure S2B), already suggesting that the increased miR-101 levels antagonize the synthesis of both proteins. And indeed, upon transfection of miR-101 inhibitor, POMP protein levels were increased in TPA-treated HepG2 cells (Figure 2D), indicating that endogenous miR-101 suppresses POMP levels. These findings strongly suggest that miR-101 indeed functions as a physiologically relevant antagonist of POMP expression. As predicted by TargetScan (http:// www.targetscan.org/), there are two miR-101 binding sites on the human POMP 30 UTR (Figure S3). We cloned the POMP 30 UTR downstream of a luciferase reporter, with or without mutations in the miR-101 binding sites. Upon transfection, miR-101 only reduced the wild-type reporter activity, to a degree that is comparable to the reduction previously observed with a similar construct containing the 30 UTR of EZH2 (Varambally et al.,

(D) Immunoblot analysis of p21, Mdm2, and p27 protein in scr- or miR-101-transfected cells (U2OS, HCT116, and MCF7). (E) qRT-PCR of p53, p21, Mdm2, and p27 mRNA expression in scr- or miR-101-transfected U2OS cells. Data are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant. (F) Cycloheximide (CHX) chase assays to determine the half-lives of p53, p21, and Mdm2 proteins following transfection of scr or miR-101. At 48 hr posttransfection, cells were treated with CHX (50 mg/ml) for the indicated time periods and then harvested for immunoblot analysis. (G) Immunoblot analysis of endogenous poly-ubiquitinated proteins in scr- or miR-101-transfected cells (U2OS and HCT116) using an anti-ubiquitin antibody.

Molecular Cell 59, 1–15, July 16, 2015 ª2015 Elsevier Inc. 3


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Figure 2. miR-101 Targets the Proteasome Maturation Protein POMP (A) Differential expression profile upon transfection with miR-101. Microarray data displayed by scatter diagram showing the relative gene expression in scrversus miR-101-transfected U2OS cells. The raw signal level for each gene is displayed in relative fluorescence units (Log2 fl). Each value represents the average of three biological replicates. (B) qRT-PCR of POMP mRNA expression in scr- or miR-101-transfected U2OS cells. Columns represent the mean ± SD of three independent experiments. (C) POMP levels decrease in miR-101-transfected U2OS, MCF7, and HCT116 cells compared to scr-transfected controls, similar to targeted knockdown of POMP. (D) POMP levels increase in miR-101 inhibitor-transfected HepG2 cells, compared to control-transfected cells. At 24 hr post-transfection, the cells were treated with 100 nM TPA for another 48 hr and then harvested for immunoblot analysis. The right panel quantitates the band intensity corresponding to POMP, normalized to the loading control. (E) Dual-luciferase reporter assays (Promega) showing repression of the wild-type POMP 30 UTR by miR-101. Mutant 30 UTRs are depicted in Figure S3. (F) Inverse correlation between miR-101 and POMP mRNA expression in ER+ breast tumor tissues, as derived from GEO: GSE29173 (Farazi et al., 2011). Data are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant. See also Figures S1–S4 and Tables S1, S2, and S3.



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Figure 3. miR-101 Interferes with Proteasome Assembly and Activity (A) In vitro proteasome activity assay by native PAGE substrate overlay showing decreased activity of both 20S and 26S proteasome activity by miR-101 or si POMP. (B) Quantitation of the in vitro proteasome activity, normalized to scr or si ctrl (n = 3). The values of scr and si ctrl were set as 100. (C) Immunoblot analysis of proteasome assembly by native PAGE and immunoblotting using an a6 antibody that stains proteasome complexes and their assembly intermediates. GAPDH serves as a loading control. (D) Protein levels of overexpressed POMP in U2OS cells that stably express POMP either with wild-type (wt) or mutant 30 UTR (mut) compared to parental cells. (E) mRNA levels of the same cells as above, as determined by qRT-PCR. (F) Levels of POMP, EZH2, and poly-ubiquitinated proteins in scr- or miR-101-transfected U2OS cells that stably express POMP either with wild-type (wt) or mutant 30 UTR (mut), as detailed in Figure S3. (G) miR-101-resistant POMP restores proper turnover of proteasome substrates in the face of forced miR-101 expression. p53, p21, Mdm2, and p27 proteins in Scr- or miR-101-transfected U2OS cells stably expressing POMP either with wild-type (wt) or mutant 30 UTR (mut) were analyzed by immunoblot. Data are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S3.



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Figure 4. miR-101 Suppresses Tumor Cell Growth and Acts as a Chemosensitizer (A and B) Cell proliferation assay following scr, miR-101, si ctrl, or si POMP transfection. The curves represent cell numbers (A) and cell confluence (B). (C) miR101-resistant POMP overexpression restores cell growth in the presence of miR-101. Cell proliferation following scr or miR-101 transfection in U2OS cells stably expressing POMP either with wild-type (wt) or mutant 30 UTR (mut) was monitored by a Celigo Cell Cytometer. (D) miR-101 arrests cells preferentially in the G2/M phase of the cell cycle. Flow cytometry analysis of U2OS cells transfected with Scr, miR-101, si ctrl, or si POMP. (E) miR-101 induces increased apoptosis. Caspase activity assay showing increased apoptosis by miR-101 or si POMP in HCT116 cells, normalized to scrtransfected cells. (legend continued on next page)



2008). In contrast, miR-101 did not significantly affect the reporter when the mutations were introduced (Figure 2E). We conclude that miR-101 directly targets POMP mRNA. Of note, an analysis of the human tumor tissue dataset GEO: GSE29173 (Farazi et al., 2011) revealed that miR-101 and POMP expression levels were inversely correlated in ER-positive breast cancers (Figure 2F; p = 0.0007). This inverse correlation between miR-101 and POMP levels was also observed between adjacent normal and malignant colonic mucosa in rectal carcinomas (Figure S4), suggesting that the POMP-antagonizing activity of miR-101 is relevant in human cancers.

enough to abolish both effects completely (Figure 3F). Importantly, another known miR-101 target, EZH2 (Varambally et al., 2008), was still reduced in its levels by miR-101, regardless of the transfected POMP construct, indicating that neither construct compromised the general activity of miR-101 in gene regulation. Moreover, we observed the accumulation of p53, p21, and p27 by miR-101 in cells that contained a POMP mRNA with wild-type 30 UTR, but again, this effect was entirely abolished when the 30 UTR was mutant (Figure 3G). In conclusion, miR-101 interferes with proteasome assembly and activity by targeting specific sites within the 30 UTR of POMP.

miR-101 Interferes with Proteasome Assembly and Activity POMP functions as an essential regulator of proteasome assembly. To test whether miR-101, via downregulation of POMP, interferes with the formation and function of proteasome, we transfected cells with miR-101 or POMP siRNA and assayed the proteasomal CT-L peptide-hydrolyzing activity in total cell lysates. Indeed, both miR-101 as well as POMP siRNA reduced proteasomal CT-L activity (Figures 3A and 3B). Consistently, we detected lower 20S and 26S proteasome levels upon transfection with miR-101 or POMP siRNA in U2OS and HeLa cells (Figure 3C). Of note, the overall proteasome levels were different between the two cell lines, consistent with previous findings that cells of various origins contain divergent amounts of proteasomes (Kumatori et al., 1990; Kanayama et al., 1991). Independent of this, however, no 16S precursor complex was formed in cells treated with miR-101 or POMP siRNA. Instead, an assembly intermediate smaller than the 16S precursor was augmented, most likely the 13S precursor complex (Figure 3C). This fits the previous observation that POMP recruits beta subunits to precursor complexes to form the 20S proteasome core complex (Fricke et al., 2007). Next, we assessed whether compromised proteasome activity in response to miR-101 is dependent on POMP suppression, and whether the interactions between miR-101 and the POMP 30 UTR are required for this. We stably transfected U2OS cells to express POMP with its wild-type 30 UTR or with a mutant 30 UTR that contains four base exchanges at each of the two miR-101-binding sites to eliminate base pairing with the miR101 seed sequence (Figure S3). The overexpression of POMP only resulted in enhanced mRNA levels, but not protein levels (Figures 3D and 3E), due to previously described feedback regulation of POMP (Heink et al., 2005). When transfected with miR101, the cells containing the wild-type construct showed severely reduced POMP levels and increased amounts of ubiquitinated proteins; in contrast, the mutation in the 30 UTR was

miR-101 Suppresses Tumor Cell Growth and Acts as a Chemosensitizer Since pharmacological proteasome inhibitors represent anti-tumor agents, and since miR-101 functions as an endogenous proteasome inhibitor, we hypothesized that the restoration of miR-101 in tumor cells suppresses cell growth. Indeed, miR101, much like POMP siRNA, strongly reduced the proliferation of various tumor cell lines (U2OS, HCT116, and MCF7) (Figures 4A, 4B, and S5A). Interestingly, miR-101 no longer suppressed the proliferation of cells stably expressing miR-101-resistant POMP with mutant 30 UTR (Figure 4C), indicating that the growth-suppressive effect of miR-101 is completely dependent on POMP suppression. As shown by flow cytometry in Figure 4D, miR-101 arrested cells preferentially in the G2/M phase of the cell cycle. A similar pattern of arrest was observed when cells were transfected with POMP siRNA (Figure 4D) or treated with pharmacological proteasome inhibitors MG-132 or bortezomib (Figure S5B). In addition, miR-101 or POMP siRNA led to an increase in caspase activity, reflecting enhanced apoptosis (Figures 4E and S5C). Furthermore, we tested whether endogenous miR-101 is involved in the regulation of cell proliferation. As shown in Figure S2A, endogenous miR-101 was induced in HepG2 cells upon TPA treatment. In addition, TPA treatment suppressed cell proliferation and induced a more differentiated cell morphology (Figures S5D and S5E). Antagonizing miR-101 restored cell growth to a significant extent (Figure 4F), whereas POMP knockdown reversed this effect (Figure 4G). This strongly suggests that endogenous miR-101 suppresses cell proliferation specifically by antagonizing POMP expression. Proteasome inhibitors are usually combined with other chemotherapeutic drugs to treat cancer. We reasoned that miR-101 may also act as a chemosensitizer. To test this, we challenged tumor cells with both miR-101 and the DNA-damaging drug camptothecin (CPT). As shown in Figures 4H and 4I, miR-101-transfected cells showed more cleaved caspase-3 and cleaved PARP in

(F) Antagonizing miR-101 restores cell growth suppression by TPA. HepG2 cells were transfected with negative control inhibitor or miR-101 inhibitor. At 24 hr post-transfection, the cells were treated with DMSO or 100 nM TPA for another 24 hr. Then, the cell numbers were determined. The cell number for the anti-NCand DMSO-treated sample was set as 100. (G) POMP knockdown reverses the effects of miR-101 inhibitor on cell growth. HepG2 cells were transfected with negative control inhibitor, miR-101 inhibitor, si ctrl, or si POMP, alone or combined. At 24 hr post-transfection, the cells were treated with 100 nM TPA for another 24 hr, followed by cell counting as in (F). (H and I) Chemosensitization by miR-101. Immunoblot analysis of cleaved caspase-3 and cleaved PARP was performed after transfecting U2OS (H) or HCT116 (I) cells with scr or miR-101. At 24 hr post-transfection, cells were treated with DMSO or CPT (200 nM, 500 nM) for another 24 hr. Data are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant. See also Figure S5.



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Figure 5. Correlation between miR-101 or POMP Expression and Survival of Breast Cancer Patients (A) Kaplan-Meier analysis showing a positive correlation between miR-101 expression and survival of ER-positive, but not ER-negative, breast cancer patients. (B) Kaplan-Meier analysis showing an inverse correlation between POMP expression and survival of ER-positive, but not ER-negative, breast cancer patients. ER status is derived from gene expression data. (legend continued on next page)



response to CPT treatment, indicating that miR-101 sensitized cells to the genotoxic drug. High Levels of miR-101 and Lower Levels of POMP Associate with Longer Survival in ER-Positive Breast Cancer Patients We next asked whether miR-101 and POMP expression levels could predict the outcome of malignant diseases. By analyzing a large gene expression and patient survival database for breast cancer (BreastMark, http://glados.ucd.ie/BreastMark/ miRNA_analysis.html) (Madden et al., 2013), we found that miR101 expression levels above the median were significantly associated with longer relapse-free survival for estrogen receptor (ER)positive breast cancer patients, but not for ER-negative breast cancer patients (Figure 5A). Furthermore, using another database (Kaplan-Meier Plotter, http://kmplot.com) (Gyo¨rffy et al., 2010), we found that high POMP expression significantly correlated with poor relapse-free survival for ER-positive, but not ER-negative, patients (Figure 5B). This suggests that miR-101 and POMP levels may predict disease progression in human breast cancer patients with positive ER status. Of note, this does not exclude the possibility that ER-negative tumors would also respond to increased miR101 or decreased POMP. The lack of a correlation between POMP and survival in these patients merely suggests that they might rely on POMP to a lesser degree, perhaps due to a more robust system of proteasome assembly. miR-101 Compromises the Estrogen Response in Breast Cancer Cells Next, we asked why miR-101 might represent a particularly important antagonist of ER-positive breast cancer. A major regulatory pathway governing ERa protein stability is the ubiquitinproteasome pathway. A number of studies have suggested that proteasome inhibition impairs ERa-dependent gene transcription (Lonard et al., 2000; Stenoien et al., 2001; Reid et al., 2003; Prenzel et al., 2011). Several mechanisms for this effect of proteasome inhibition have been proposed, including decreased ERa mobility in the nucleus (Stenoien et al., 2001; Reid et al., 2003), decreased ERa binding and recruitment of cofactors (Reid et al., 2003), and impaired transcriptional elongation on estrogen target genes due to decreased histone H2B mono-ubiquitination (Prenzel et al., 2011). Thus, we hypothesized that miR-101 expression or POMP knockdown may impair ERa-dependent gene transcription. To test this, we transfected the ER-positive breast cancer cell line MCF-7 with miR-101 or POMP siRNA and treated the cells with or without 17b-estradiol (E2). Three ERa-dependent genes (CXCL12, GREB1, and PGR) were used to monitor the estrogen response. Indeed, in the presence of E2, their induction was compromised by miR-101 to a similar degree as by POMP siRNA (Figures 5C and 5D). Clonogenic assays also revealed that miR-101 or POMP siRNA reduced the estrogen-driven proliferation of MCF7 cells (Figures

5E and 5F). We conclude that miR-101, by targeting POMP, interferes with estrogen-induced gene expression, leading to compromised proliferation of ER-positive breast cancer cells. POMP Suppression Interferes with Breast Cancer Growth and Triggers Tumor Regression Since miR-101 has been shown to suppress tumor growth in vivo (Varambally et al., 2008; Su et al., 2009; Yan et al., 2014), we tested whether POMP downregulation may represent a plausible mechanism. First, we engineered MCF7 cells with doxycycline (Dox)-inducible expression of POMP short hairpin RNA (shRNA) (Figure 6A). In this system, Dox strongly decreased mRNA levels of POMP (Figure S6A), resulting in accumulation of p53 and cell growth defects (Figures S6B and S6C). We injected these MCF7 cells subcutaneously into the left and right flank of immunodeficient female mice (Figure 6B). As shown in Figure 6C, the size of the tumors on both flanks was equal in the absence of Dox. In the presence of Dox, however, cells harboring POMP shRNA formed tumors initially, but these tumors failed to grow further and became almost invisible after 5 weeks (Figure 6D). This strongly suggests that POMP inhibition suppresses breast tumor progression. To test if POMP inhibition triggers tumor regression, we started to feed mice with Dox starting at week 5 after injection of MCF7 cells, when the tumors were well established. Indeed, the tumors harboring POMP shRNA no longer grew, but instead regressed to barely detectable volumes, while control tumors grew robustly (Figure 6E). At week 10, the remaining tumors were extracted for further analysis. We confirmed downregulation of POMP mRNA by POMP shRNA in the tumor samples (Figure 6F), along with accumulated p53 and p21 proteins (Figure 6G) and significantly lower numbers of Ki67-positive nuclei, but increased cleaved caspase-3 (Figures 6H and 6I), indicating that POMP knockdown also suppresses cell proliferation and induces apoptosis in vivo. POMP Suppression Overcomes Tumor Cell Resistance to Bortezomib Unlike the proteasome inhibitor bortezomib, which directly targets proteolytic sites within the proteasome, POMP inhibition interferes with proteasome functions by disrupting its assembly. We therefore asked whether POMP inhibition is capable of overcoming resistance to bortezomib. Bortezomib-resistant U2OS cells were established by a stepwise increase of the concentration of bortezomib to 12.5 nM (Fuchs et al., 2008). This concentration of bortezomib corresponds to plasma concentrations measured in patients during bortezomib therapy (Papandreou et al., 2004). As shown in Figure 7A, the resistant U2OS cells grew in the presence of 12.5 nM bortezomib, whereas parental cells stopped growing. Even at higher concentrations of bortezomib, the resistant cells showed a strongly diminished apoptotic response (Figure 7B). Accordingly, the resistant cells

(C and D) qRT-PCR analyzing the expression of representative estrogen-dependent genes (CXCL12, GREB1, and PGR) and POMP in the absence or presence of E2 in scr- or miR-101-transfected MCF7 cells (C) or si ctrl- or si POMP-transfected MCF7 cells (D). (E and F) Colony formation assays showing estrogen-dependent and -independent cell growth suppression by miR-101 expression (E) and POMP knockdown (F) in MCF7 cells. The right panels quantitate the colony numbers, normalized to untreated scr- or si ctrl-transfected cells. Data are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.



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Figure 6. POMP Inhibition Suppresses Breast Cancer Growth and Triggers Tumor Regression (A) Schematic diagram showing the TRIPZ-inducible shRNA expression system used for expressing POMP shRNA in vivo. (B) Schematic diagram of subcutaneous cell injections into the flanks of female nude mice. The left flank received ctrl shRNA cells (shctrl), and the right flank received POMP shRNA cells (shPOMP). (C and D) Xenograft experiments showing suppressed tumor growth by POMP inhibition. Female nude mice were injected subcutaneously into the flanks as indicated in (B). At 3 days prior to injection, mice were administered 17b-estradiol (0,67 mg/ml) via drinking water containing 5% sucrose with or without Dox (1 mg/ml). Graph represents the mean of tumor volumes (mm3) ± SD. (E) Xenograft experiments showing tumor regression by POMP inhibition. Female nude mice were estradiol-treated and injected as in (B). At 5 weeks post-injection, we began to treat the mice with Dox in the drinking water (1 mg/ml). (F) qRT-PCR showing POMP expression in harvested tumors at endpoint (9 weeks, 4 tumors each for shctrl and shPOMP) from (E). (legend continued on next page)



accumulated far less poly-ubiquitinated proteins when treated with bortezomib, compared to parental cells (Figure 7C). Previous studies suggested that cells acquire resistance to proteasome inhibitors by increasing proteasome subunit levels and proteasome biogenesis (Meiners et al., 2003; Fuchs et al., 2008; Radhakrishnan et al., 2010; Steffen et al., 2010). Indeed, we found that the resistant cells contained more 20S proteasome core subunits (Figure 7D). Next we tested whether miR101, via targeting POMP, could overcome the resistance to bortezomib. Indeed, miR-101 overexpression as well as POMP knockdown by siRNA accumulated poly-ubiquitinated proteins in bortezomib-resistant cells (Figure 7E). Thus, miR-101 is still able to interfere with proteasome functions in bortezomib-resistant cells. Moreover, resistant cells no longer proliferated upon miR-101 expression or POMP knockdown by siRNA (Figure 7F) due to G2/M arrest (Figure 7G). Finally, as shown in Figure 7H, treating resistant cells with either miR-101 or POMP siRNA or 12.5 nM bortezomib did not induce apoptosis, but the combined treatment did. Similar re-sensitizing effects were observed when analyzing MCF7 cells (Figure S7). Thus, suppressing POMP levels re-sensitizes resistant cancer cells to bortezomib. DISCUSSION Our study uncovers an unexpected role of miR-101 as an endogenous proteasome inhibitor by targeting the proteasome biogenesis. The inhibitory function of miR-101 becomes evident by a prolonged half-life of proteasomal substrates and the accumulation of ubiquitin conjugates. We identified the proteasome maturation protein POMP as a target of miR-101 and as the central mechanism by which miR-101 mediates proteasome inhibition. In vitro and in vivo experiments indicated the robust anti-proliferative effect of POMP knockdown in cancer cells. Importantly, miR-101-inhibited cancer cell proliferation can be completely rescued by POMP overexpression. Taken together, these results indicate that miR-101 acts as an endogenous proteasome inhibitor by targeting POMP, and that POMP targeting is necessary and sufficient to suppress tumor proliferation. For an miRNA acting as a regulator, a limited half-life of POMP is necessary. And indeed, it was previously found that POMP has a biological half-life of less than 2 hr (Heink et al., 2005). Moreover, the proteasome itself, despite having long-lived components (Hendil, 1988; Tanaka and Ichihara, 1989), requires constant assembly to maintain its function, i.e., for attachment of the 19S regulator to the 20S core unit (Sasaki et al., 2010), for immunoproteasome function (Heink et al., 2005), for nuclear transport (Reits et al., 1997), and for the generation of new proteasomes in rapidly dividing cells. Because proteasome biogenesis requires the coordinated expression of all genes encoding proteasome subunits and assembly factors, it is conceivable that these genes share common

regulatory elements. Indeed, in mammalian cells, almost all genes encoding proteasome subunits as well as POMP contain a common regulatory motif termed AREs (antioxidant response elements) (Steffen et al., 2010). TCF11/Nrf1 (TCF11 is the longer isoform of Nrf1 in humans) and Nrf2 have been reported to upregulate the expression of proteasome subunits and POMP by binding to the ARE in response to proteasome inhibition (Kwak et al., 2003; Radhakrishnan et al., 2010; Steffen et al., 2010). In addition to the transcriptional level, our study demonstrates that POMP expression can also be regulated post-transcriptionally by miR-101. Importantly, we found an inverse correlation between miR-101 and POMP expression levels in breast and rectal cancer tissues, suggesting that the miR-101-POMP axis may also determine the fate of human cancer cells in vivo. Indeed, we observed higher POMP levels and lower amounts of miR101 in rectal cancers compared to normal adjacent tissues (Figure S4B). This might be one mechanism of how cancer cells acquire increased proteasome levels and activities. Based on the role of miR-101 in interfering with proteasome biogenesis, this study outlines a possible future strategy to target proteasome functions. So far, efforts of drug discovery to manipulate the UPS mainly focused on identifying small molecules targeting proteolytic sites of the proteasome or ubiquitin pathways upstream of the proteasome. In contrast, therapeutic interventions to interfere with proteasome biogenesis have been largely neglected. The rationale to target proteasome biogenesis is consistent with enhanced proteasome biogenesis in cancer cells (Chen and Madura, 2005; Bazzaro et al., 2006). Ubiquitinated proteins are detected at high levels in cancer cells (Chen and Madura, 2005; Bazzaro et al., 2006), indicating that more misfolded and damaged proteins accumulate in them. Due to a higher demand of proteolytic activities, cancer cells seem to increase proteasome biogenesis to cope with proteolytic stress. Specifically, the reported transcription factors (TCF11/Nrf1 and Nrf2) that promote the synthesis of proteasome subunits and assembly factors all respond to oxidative stress, a frequent condition in cancer cells (Toyokuni et al., 1995). In response to proteasome inhibitors, cancer cells use autoregulatory feedback mechanisms to increase proteasome subunit expression and proteasome biogenesis, thus reducing drug efficacy (Meiners et al., 2003; Fuchs et al., 2008). Interfering with proteasome assembly would overcome this resistance. Indeed, we observed that bortezomib-resistant cells are still sensitive to POMP suppression. In addition to targeting the proteasome, miR-101 has been reported to act as a potential inhibitor of autophagy by targeting RAB5A and ATG4B, which support autophagosome formation (Frankel et al., 2011). Although proteasomal degradation and autophagy represent distinct pathways, their regulation involves cross-talk (Korolchuk et al., 2010). Impaired proteasome function can lead to increased autophagy function (Iwata et al.,

(G) Accumulation of p53 and p21 in shPOMP-expressing tumors. Representative immunofluorescence image; scale bar, 50 mm. (H) Reduced Ki67-positive cells in shPOMP-expressing tumors. Upper, representative immunohistochemical image; scale bar, 50 mm. Lower, the percentage of Ki67-positive cells was counted per high-power field (4003), three fields per tumor (n = 6). (I) Accumulated cleaved caspase-3 in shPOMP-expressing tumors. Representative immunohistochemical image; scale bar, 50 mm. Data are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S6.



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Figure 7. POMP Suppression Overcomes Tumor Cell Resistance to Bortezomib (A) Proliferation of parental and bortezomib-resistant U2OS cells in the presence of 12.5 nM bortezomib. The curves represent cell numbers. (B) Resistant cells undergo less apoptosis upon bortezomib. Immunoblot analysis of PARP cleavage for parental and resistant U2OS cells after treatment with bortezomib at the indicated concentrations for 48 hr. (C) Resistant cells accumulate less poly-ubiquitinated proteins in response to bortezomib. Immunoblot analysis of poly-ubiquitinated proteins from parental and resistant U2OS cells after treatment with 20 nM bortezomib for the indicated times. (D) Levels of 20S proteasome a/b subunits in parental and bortezomib-resistant U2OS cells. (legend continued on next page)



2005; Pandey et al., 2007). This suggests that eukaryotic cells evolved a compensatory mechanism, using autophagy to reduce the burden of accumulated substrates of the proteasome. Thus, proteasome inhibitor-based therapy can fail due to enhanced autophagy. It will be of interest to evaluate how miR-101 regulates both intracellular protein degradation pathways (proteasomal degradation and autophagy) and their cross-talk. As a tumor-suppressive miRNA, the miR-101 gene locus was observed to be deleted in some tumors (Varambally et al., 2008). However, epigenetic silencing of miR-101 was also found in bladder cancer and hepatocellular carcinoma (Kottakis et al., 2011; Wang et al., 2014). The epigenetic silencing of miR-101 involves EZH2, which binds to the miR-101 promoter and negatively regulates its expression. Interestingly, as EZH2 is also a target of miR-101, this suggests a double-negative feedback loop between miR-101 and EZH2. Thus, pharmacological inhibition of EZH2 by recently developed small molecules (Tan et al., 2007) may reactivate miR-101 causing growth suppression in cancer cells. In addition, treatment of hepatoma HepG2 cells by TPA upregulates miR-101 through enhanced MAP kinase activity (Chiang et al., 2010). In this study, we confirmed this result and also found that miR-101 contributes to TPA-suppressed cell growth. Thus, restoration of miR-101 expression in tumor cells represents a viable option to decrease proteasome activity and interfere with tumor progression. EXPERIMENTAL PROCEDURES For detailed descriptions, please refer to the Supplemental Experimental Procedures. Cell Culture and Transfection U2OS, HeLa, and HepG2 cells were maintained in DMEM and HCT116 cells in McCoy’s medium, each with 10% FCS. MCF7 cells were maintained in phenol red-free DMEM with high glucose (4.5 g/l). Transfections were performed using Lipofectamine 2000 (Invitrogen). qRT-PCR Analysis and Microarray Analysis Total RNA including miRNA was isolated using TRIzol (Invitrogen). The relative mRNA levels were determined by reverse transcription and qPCR with Sybr Green dye (Invitrogen). Mature miR-101 was quantified using the TaqMan MicroRNA Assays (Applied Biosystems). To determine gene expression profiles affected by miR-101 in U2OS cells, one-color microarray was performed (Agilent Technologies). Animal Studies All animal works were carried out in full agreement with the Go¨ttingen University Animal Care Committee and the Institutional Guidelines for Humane Use of Animals in Research. For subcutaneous injection, 5 3 106 MCF7 shctrl or shPOMP cells were injected into the left and right flanks of the female SCIDSHO mice. Tumor growth was monitored once a week.

Statistical Analysis Data are presented as mean ± SD of at least three independent experiments. Student’s t test was used for comparisons. p values < 0.05 were considered to reflect statistical significance. ACCESSION NUMBERS The accession number for the raw microarray data reported in this paper is GEO: GSE69150. SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures, three tables, and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.molcel.2015.05.036. AUTHOR CONTRIBUTIONS M.D. and X.Z. conceived and designed the experiments; X.Z., R.S., S.E., E.K., and E.C.-B. performed the experiments; E.M., A.J.L., and T.B. performed bioinformatics analyses; J.G. and M.G. provided human CRC data; U.M.M. provided support for the xenograft experiment; M.D. raised funding for the study; and X.Z. and M.D. wrote the paper. ACKNOWLEDGMENTS Our work was supported by the German Cancer Aid/Dr. Mildred Scheel Stiftung, the Wilhelm Sander Stiftung, the Deutsche Jose´ Carreras Stiftung, the Else Kro¨ner-Fresenius Stiftung, and the German Research Foundation. E.K. was supported by the SFB740. Daniela Ludwig is acknowledged for her excellent technical assistance. X.Z. was supported by the Go¨ttingen Graduate School of Neurosciences and Molecular Biosciences (GGNB), funding line 1 of the German Excellence Initiative. Received: February 2, 2015 Revised: May 4, 2015 Accepted: May 26, 2015 Published: July 2, 2015 REFERENCES Bazzaro, M., Lee, M.K., Zoso, A., Stirling, W.L., Santillan, A., Shih, IeM., and Roden, R.B. (2006). Ubiquitin-proteasome system stress sensitizes ovarian cancer to proteasome inhibitor-induced apoptosis. Cancer Res. 66, 3754– 3763. Blagosklonny, M.V., Wu, G.S., Omura, S., and el-Deiry, W.S. (1996). Proteasome-dependent regulation of p21WAF1/CIP1 expression. Biochem. Biophys. Res. Commun. 227, 564–569. Buac, D., Shen, M., Schmitt, S., Kona, F.R., Deshmukh, R., Zhang, Z., Neslund-Dudas, C., Mitra, B., and Dou, Q.P. (2013). From bortezomib to other inhibitors of the proteasome and beyond. Curr. Pharm. Des. 19, 4025–4038. Burri, L., Ho¨ckendorff, J., Boehm, U., Klamp, T., Dohmen, R.J., and Le´vy, F. (2000). Identification and characterization of a mammalian protein interacting with 20S proteasome precursors. Proc. Natl. Acad. Sci. USA 97, 10348– 10353.

(E) miR-101 or POMP knockdown by siRNA accumulates poly-ubiquitinated proteins in bortezomib-resistant cells. Immunoblot analysis of poly-ubiquitinated proteins from resistant U2OS cells transfected with miR-101 or si POMP twice for 3 days. (F) Resistant cells no longer proliferate upon miR-101 expression or POMP knockdown. Proliferation of resistant U2OS cells transfected with si ctrl or si POMP. The curves represent cell numbers. (G) miR-101 expression or POMP knockdown arrests cells in the G2/M phase in resistant cells. Flow cytometry of resistant U2OS cells, transfected with miR-101 or si POMP twice for 3 days. (H) Combined treatment of miR-101 expression or POMP knockdown and bortezomib induces apoptosis in resistant cells. PARP cleavage after transfection with miR-101 or si POMP for 2 days, treated with or without 12.5 nM bortezomib. Data are presented as mean ± SD of three independent experiments. See also Figure S7.



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A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases.

Shikonin Promotes Skin Cell Proliferation and Inhibits Nuclear Factor-κB Translocation via Proteasome Inhibition In Vitro.

Platinum-containing compound platinum pyrithione suppresses ovarian tumor proliferation through proteasome inhibition.

Targeting the proteasome in epilepsy.

An oral proteasome inhibitor for multiple myeloma.

Long-range allosteric regulation of the human 26S proteasome by 20S proteasome-targeting cancer drugs.

Proteasome inhibitor patents (2010 - present).

The resistance mechanisms of proteasome inhibitor bortezomib.

An inducible chaperone adapts proteasome assembly to stress.

Melatonin as a proteasome inhibitor. Is there any clinical evidence?

Ubiquitinated proteasome inhibitor is a component of the 26 S proteasome complex.

Precise assembly and regulation of 26S proteasome and correlation between proteasome dysfunction and neurodegenerative diseases.

GSK1838705A, an IGF-1R inhibitor, inhibits glioma cell proliferation and suppresses tumor growth in vivo.

Suppression of the Oncogenic Transcription Factor FOXM1 by Proteasome Inhibitors.

Proteasome inhibitor bortezomib suppresses nuclear factor-kappa B activation and ameliorates eye inflammation in experimental autoimmune uveitis.

MicroRNA-195 suppresses tumor cell proliferation and metastasis by directly targeting BCOX1 in prostate carcinoma.

MicroRNA-361-3p suppresses tumor cell proliferation and metastasis by directly targeting SH2B1 in NSCLC.

The mechanism for molecular assembly of the proteasome.

MicroRNA-188-5p suppresses tumor cell proliferation and metastasis by directly targeting FGF5 in hepatocellular carcinoma.

Proteasome inhibitor MG132 inhibits the proliferation and promotes the cisplatin-induced apoptosis of human esophageal squamous cell carcinoma cells.

Upregulation of proteasome activity rescues cardiomyocytes following pulse treatment with a proteasome inhibitor.

Alpha-ring Independent Assembly of the 20S Proteasome.

Synergistic targeting of Sp1, a critical transcription factor for myeloma cell growth and survival, by panobinostat and proteasome inhibitors.

Architecture and assembly of the archaeal Cdc48*20S proteasome.