Ribosomopathy-like properties of murine and human cancers

<div><p>Ribosomopathies comprise a heterogeneous group of hematologic and developmental disorders, often characterized by bone marrow failure, skeletal and other developmental abnormalities and cancer predisposition. They are associated with mutations and/or haplo-insufficiencies of ribosomal proteins (RPs) and inefficient ribosomal RNA (rRNA) processing. The resulting ribosomal stress induces the canonical p19^<i>ARF</i>/Mdm2/p53 tumor suppressor pathway leading to proliferative arrest and/or apoptosis. It has been proposed that this pathway is then inactivated during subsequent neoplastic evolution. We show here that two murine models of hepatoblastoma (HB) and hepatocellular carcinoma (HCC) unexpectedly possess features that mimic the ribosomopathies. These include loss of the normal stoichiometry of RP transcripts and proteins and the accumulation of unprocessed rRNA precursors. Silencing of p19^<i>ARF</i>, cytoplasmic sequestration of p53, binding to and inactivation of Mdm2 by free RPs, and up-regulation of the pro-survival protein Bcl-2 may further cooperate to drive tumor growth and survival. Consistent with this notion, re-instatement of constitutive p19^<i>ARF</i> expression in the HB model completely suppressed tumorigenesis. In >2000 cases of human HCC, colorectal, breast, and prostate cancer, RP transcript deregulation was a frequent finding. In HCC and breast cancer, the severity of this dysregulation was associated with inferior survival. In HCC, the presence of RP gene mutations, some of which were identical to those previously reported in ribosomopathies, were similarly negatively correlated with long-term survival. Taken together, our results indicate that many if not all cancers possess ribosomopathy-like features that may affect their biological behaviors.</p></div

Perturbation of the c‑Myc–Max Protein–Protein Interaction via Synthetic α‑Helix Mimetics

The rational design of inhibitors of the bHLH-ZIP oncoprotein c-Myc is hampered by a lack of structure in its monomeric state. We describe herein the design of novel, low-molecular-weight, synthetic α-helix mimetics that recognize helical c-Myc in its transcriptionally active coiled-coil structure in association with its obligate bHLH-ZIP partner Max. These compounds perturb the heterodimer’s binding to its canonical E-box DNA sequence without causing protein–protein dissociation, heralding a new mechanistic class of “direct” c-Myc inhibitors. In addition to electrophoretic mobility shift assays, this model was corroborated by further biophysical methods, including NMR spectroscopy and surface plasmon resonance. Several compounds demonstrated a 2-fold or greater selectivity for c-Myc–Max heterodimers over Max–Max homodimers with IC₅₀ values as low as 5.6 μM. Finally, these compounds inhibited the proliferation of c-Myc-expressing cell lines in a concentration-dependent manner that correlated with the loss of expression of a c-Myc-dependent reporter plasmid despite the fact that c-Myc–Max heterodimers remained intact

RP transcript deregulation in human cancers.

<p>3D area maps of transcript levels for 77 RPs expressed in HCCs (A), CRCs (B), BCs (C) and PCs (D). To better evaluate differences in the other transcripts which did reach significance for their F-tests, these transcripts–<i>Rps26</i>, <i>Rpl9</i>, <i>Rps27</i>, <i>Rps28</i>, and <i>Rpl21</i>– were excluded from 3D area plots. For each cancer, tumors with matched samples of normal tissue in TCGA were selected for direct comparison (50 for HCC, 41 for CRC, 113 for BC and 52 for PC). Relative expression for each RP transcript was calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.g001" target="_blank">Fig 1</a>. See Figures D-G in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.s001" target="_blank">S1 File</a> for 3D area plots of the above matched tumor data together with additional data from unmatched tumor samples. (E, F) Patient survival in HCC and BC inversely correlates with the severity of RP transcript deregulation. Patients were sorted according to their RP transcript deregulation, and survival curves were plotted for the top and bottom 25% of patients with the greatest and least degree of RP transcript deregulation.</p

Incomplete processing of rRNAs in HBs and HCCs.

<p>(A) Normal rRNA processing. Arrows depict regions amplified by qRT-PCR to quantify 18S-ITS1, ITS1-5.8S, 5.8S-ITS2 and ITS2-28S junctional fragments common to all rRNA precursors. (B) Quantification of each of the above four junctions in control livers and HBs. Identically colored dots represent the same tumor RNA sample within the subgroup of tumors that demonstrated abnormal processing of at least one junction. Control livers and tumors with no significant processing differences are depicted in black. (C) Similar quantification of RNA processing in HCCs. Data in (B) and (C) were normalized to levels of total 18S and 28S RNA. Each qRT-PCR reaction was performed in triplicate and the mean is depicted.</p

Relative RP transcript and protein levels differ in murine models of HB and HCC.

<p>(A) RP transcript abundance in hepatocytes (H) versus HB tumors (T). Heat maps are based on averaged RNA-seq data from 4–5 samples in each group with the most abundant transcripts being shown in orange and the least abundant transcripts being shown in blue, with the sum of all transcripts in each group equaling 100%. All transcript levels are expressed as a percent of total, displayed relative to those in WT hepatocytes and do not take into account the fact, as previously shown, that average RP transcript expression was increased 5.2-fold in HBs relative to hepatocytes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.ref016" target="_blank">16</a>] *: deregulated transcripts in WT tumors vs. WT hepatocytes, ^: deregulated transcripts in KO tumors vs. KO hepatocytes. (B) RP transcript deregulation among WT and KO hepatocytes and HBs. (C) Similar heat maps from livers or HCCs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.ref013" target="_blank">13</a>]. L: control livers. 3D and 7D: livers obtained 3 and 7 days after removing doxycycline to induce Myc expression. T: initial tumors. 3R and 7R: regressing tumors following doxycycline resumption for 3 or 7 days, respectively. Additional tumor-bearing mice were maintained on doxycycline for 2.5–3 months to allow for complete regression. Doxycycline removal in these mice led to development of recurrent tumors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.ref012" target="_blank">12</a>]. *: significant differences in relative expression compared to normal liver; ^: significant differences between recurrent tumor and liver (q-value < 0.05). Relative transcript abundance was expressed as described for panel A and compared with the relative abundance in control livers. (D) RP transcript discordances between HBs and HCCs. “Opposing” directionality occurred when an HB transcript’s direction of change relative to hepatocytes differed between WT and KO HBs. (E) Immunoblots of RPs in WT and KO livers (L) and HB tumors (T). (F) Immunoblots of RPs from livers, collected as described in (C).</p

Reprograming of survival and apoptosis pathways in HBs and HCCs.

<p>(A) Expression of p19^ARF, MDM2 and p53 in total liver (L) and HB (T) lysates from WT and KO mice. (B) Similar immuno-blots from HCCs. (C) Immuno-staining of frozen sections of liver (L) and WT HBs (T) for p53 and MDM2. Using ImageJ software (<a href="https://imagej.nih.gov/ij/" target="_blank">https://imagej.nih.gov/ij/</a>), we determined that >80% of Mdm2 and p53 localized to the cytoplasm in both livers and tumors. (D) p53 and MDM2 co-localize to HB cytoplasm. A freshly collected WT HB tumor was fractionated into cytoplasmic, nuclear and nucleolar compartments. Each fraction was tested for the protein markers localizing to these compartments (GAPDH, histone H3 and fibrillarin, respectively) and in parallel for p53, p19^ARF and MDM2. Varying amounts of lysate and exposure times were required to compensate for differential protein expression. (E) Liver and HB cytoplasmic fractions were immuno-precipitated with control IgG or anti-MDM2 IgG. Precipitates were resolved by SDS-PAGE and silver stained. Bracketed regions were excised from lanes 2 and 4 and subjected to trypsin digestion and mass spectrometry. (F) Bcl-2 and Bax expression in mitochondria from WT or KO HBs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.ref016" target="_blank">16</a>]. The same blot was probed with an antibody for the mitochondrial protein pyruvate dehydrogenase E1α subunit (PDH) as a control for protein loading. The mean up-regulation of Bcl-2 relative to that in livers was 3.8-fold in WT HBs and 2.3-fold in KO HBs. The mean up-regulation of Bax was 11.7-fold in WT HBs and 10.5-fold in KO HBs. (G) Bcl-2 and Bax expression in isolated mitochondria from livers (L), tumors (T) and recurrent HCC tumors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.ref012" target="_blank">12</a>]. The mean up-regulation of Bcl-2 was 5.5-fold in initial tumors and 6.3-fold in recurrent tumors. Similarly, the mean up-regulation of Bax was 15-fold in initial tumors and 15.6-fold in recurrent tumors.</p

Model depicting the relationship between Myc and AMPK (yellow boxes) demonstrating their influence over common metabolic functions, although sometimes in opposite ways and for different purposes.

<p>Communication between Myc and AMPK may occur via at least 2 distinct and semi-autonomous routes with different initiating events and consequences. The first involves the activation of AMPK via Myc-mediated depletion of cellular ATP stores arising as a consequence of energy-consuming anabolic processes such as proliferation (-ATP, blue box) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134049#pone.0134049.g001" target="_blank">Fig 1D</a> and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134049#pone.0134049.ref023" target="_blank">23</a>]). The second involves AMPK activation via ROS generated from increases in mitochondrial metabolism or cytoplasmic signaling pathways. It is notable that, in the first case, AMPK activation is dependent upon ATP depletion whereas, in the second case, AMPK activation occurs regardless of ATP status. AMPK activation via ROS can thus anticipate impending ATP depletion and prevent or limit this by down-regulating ATP dependent processes. In the face of a pre-existing ATP deficit, other functions of AMPK such as the promotion of proliferative arrest might tend to override the effects of Myc over-expression. In contrast, activation of AMPK by ROS might reinforce an already highly proliferative and ATP-replete state by promoting pro-anabolic functions such as glycolysis and Oxphos without necessarily compromising proliferation.</p

Metabolite profiling of WT and KO MEFs.

<p>(A) HPLC-ESI-MS/MS quantification of select metabolites in MEFs prior to or following MycER activation for 8 days. Each box represents the average of biological quadruplicate samples. Levels of each depicted metabolite were arbitrarily set to 1 in WT cells (white boxes). (B) Enzymatic and metabolite feedback control of PDH and PK. Note the control of the former by the stimulatory phosphatase PDP2 and the inhibitory kinase PDK1 as well as additional indirect and direct control of PDH and PK, respectively by ATP and ADP. PDH and PK enzyme activities (C and D, respectively) and acetyl CoA assays (E) were performed on whole cell extracts as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134049#pone.0134049.ref023" target="_blank">23</a>].</p

Mitochondrial proteomic profiling.

<p>LC-MS/MS reliably identified and quantified 345 proteins with mitochondrial localization from representative peptides in each of the 4 experimental groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134049#pone.0134049.s010" target="_blank">S3 Table</a>). (A) Differential protein expression between WT and KO MEFs prior to MycER activation. (B) Differential protein expression in WT MEFs prior to or following an 8 day period of MycER activation. (C) Venn diagram of protein overlap between WT and KO MEFs, WT vs. WT+Myc and KO versus KO+Myc. Each of the protein subsets within these 3 groups are enclosed by red, green and blue circles and correspond to the proteins denoted in panels A, B and D, respectively. All proteins were selected by a conservative q-value of <0.05 by 2-way analysis of variance (two way ANOVA). (D) Differential protein expression in KO MEFs prior to or following 8 days of Myc over-expression. (E) Quantification of 8 proteins that showed a significant interaction effect by both AMPK and Myc overexpression.</p

Structural and functional properties of ETCs complexes in WT and KO cells.

<p><i>(A)</i> Analysis of ETC complexes by non-denaturing BNGE. Mitochondria were purified from triplicate cultures of the indicated cell types and resolved by BNGE. At least 5 individual supercomplexes (SC_a-e) could be resolved [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134049#pone.0134049.ref020" target="_blank">20</a>]. Note that Complex V (ATP synthase) is composed of both monomers (V_m) and dimers (V_d). <i>(B) In situ</i> enzymatic assays for Complex I (NADH ubiquinone oxidoreductase) plus supercomplexes, Complex V_m and V_d (ATPase), Complex III (decylubiquinol cytochrome c oxidoreductase) and Complex IV (Cytochrome c oxidase). Each assay was performed in triplicate on at least 2 occasions, with representative results being shown here (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134049#pone.0134049.s002" target="_blank">S2 Fig</a>). <i>(C)</i> Complex II (succinate dehydrogenase) activity was quantified on lysates prepared from purified mitochondria as its <i>in situ</i> assessment was found to be unreliable. The results shown in the histogram represent the mean of triplicate enzymatic determinations ± 1 SEM. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134049#pone.0134049.s003" target="_blank">S3 Fig</a> for quantification of all enzymatic measurements.</p