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Molecular Biology of the Cell

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Molecular Cancer Research

Protein–protein interactions (PPI) are a hallmark of cellular signaling. Such interactions occur abundantly within the cellular milieu and encompass interactions involved in vital cellular processes. Understanding the various types, mechanisms, and consequences of PPIs with respect to cellular signaling and function is vital for targeted drug therapy. Various types of small-molecule drugs and targeted approaches to drug design have been developed to modulate PPIs. Peptidomimetics offer an exciting class of therapeutics as they can be designed to target specific PPIs by mimicking key recognition motifs found at critical points in the interface of PPIs (e.g., hotspots). In contrast to peptides, peptidomimetics do not possess a natural peptide backbone structure but present essential functional groups in a required three-dimensional pattern complimentary to the protein-binding pocket. This design feature overcomes many limitations of peptide therapeutics including limited stability toward peptidases, poor transport across biologic membranes, and poor target specificity. Equally important is deciphering the structural requirements and amino acid residues critical to PPIs. This review provides an up-to-date perspective of the complexity of cellular signaling and strategies for targeting PPIs in disease states, particularly in cancer, using peptidomimetics, and highlights that the rational design of agents that target PPIs is not only feasible but is of the utmost clinical importance. Mol Cancer Res; 12(7); 967–78. ©2014 AACR.


Thyroid malignancies are the most common type of endocrine tumors. Of the various histologic subtypes, anaplastic thyroid carcinoma (ATC) represents a subset of all cases but is responsible for a significant proportion of thyroid cancer-related mortality. Indeed, ATC is regarded as one of the more aggressive and hard to treat forms of cancer. To date, there is a paucity of relevant model systems to critically evaluate how the signature genetic abnormalities detected in human ATC contribute to disease pathogenesis. Mutational activation of the BRAF protooncogene is detected in approximately 40% of papillary thyroid carcinoma (PTC) and in 25% of ATC. Moreover, in ATC, mutated BRAF is frequently found in combination with gain-of-function mutations in the p110 catalytic subunit of PI3'-Kinase (PIK3CA) or loss-of-function alterations in either the p53 (TP53) or PTEN tumor suppressors. Using mice with conditional, thyrocyte-specific expression of BRAFV600E, we previously developed a model of PTC. However, as in humans, BRAFV600E-induced mouse PTC is indolent and does not lead to rapid development of end-stage disease. Here, we use mice carrying a conditional allele of PIK3CA to demonstrate that, although mutationally activated PIK3CAH1047R is unable to drive transformation on its own, when combined with BRAFV600E in thyrocytes, this leads to development of lethal ATC in mice. Combined, these data demonstrate that the BRAFV600E cooperates with either PIK3CAH1074R or with silencing of the tumor-suppressor PTEN, to promote development of anaplastic thyroid carcinoma.

Implications: This genetically relevant mouse model of ATC will be an invaluable platform for preclinical testing of pathway-targeted therapies for the prevention and treatment of thyroid carcinoma. Mol Cancer Res; 12(7); 979–86. ©2014 AACR.


Glioblastoma multiforme (GBM) is a highly malignant human brain neoplasm with limited therapeutic options. GBMs display a deregulated apoptotic pathway with high levels of the antiapoptotic Bcl-2 family of proteins and overt activity of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Therefore, combined interference of the PI3K pathway and the Bcl-2 family of proteins is a reasonable therapeutic strategy. ABT-263 (Navitoclax), an orally available small-molecule Bcl-2 inhibitor, and GDC-0941, a PI3K inhibitor, were used to treat established glioblastoma and glioblastoma neurosphere cells, alone or in combination. Although GDC-0941 alone had a modest effect on cell viability, treatment with ABT-263 displayed a marked reduction of cell viability and induction of apoptotic cell death. Moreover, combinatorial therapy using ABT-263 and GDC-0941 showed an enhanced effect, with a further decrease in cellular viability. Furthermore, combination treatment abrogated the ability of stem cell–like glioma cells to form neurospheres. ABT-263 and GDC-0941, in combination, resulted in a consistent and significant increase of Annexin V positive cells and loss of mitochondrial membrane potential compared with either monotherapy. The combination treatment led to enhanced cleavage of both initiator and effector caspases. Mechanistically, GDC-0941 depleted pAKT (Serine 473) levels and suppressed Mcl-1 protein levels, lowering the threshold for the cytotoxic actions of ABT-263. GDC-0941 decreased Mcl-1 in a posttranslational manner and significantly decreased the half-life of Mcl-1 protein. Ectopic expression of human Mcl-1 mitigated apoptotic cell death induced by the drug combination. Furthermore, GDC-0941 modulated the phosphorylation status of BAD, thereby further enhancing ABT-263–mediated cell death.

Implications: Combination therapy with ABT-263 and GDC-0941 has novel therapeutic potential by specifically targeting aberrantly active, deregulated pathways in GBM, overcoming endogenous resistance to apoptosis. Mol Cancer Res; 12(7); 987–1001. ©2014 AACR.


Although modern radiotherapy technologies can precisely deliver higher doses of radiation to tumors, thus, reducing overall radiation exposure to normal tissues, moderate dose, and normal tissue toxicity still remains a significant limitation. The present study profiled the global effects on transcript and miR expression in human coronary artery endothelial cells using single-dose irradiation (SD, 10 Gy) or multifractionated irradiation (MF, 2 Gy x 5) regimens. Longitudinal time points were collected after an SD or final dose of MF irradiation for analysis using Agilent Human Gene Expression and miRNA microarray platforms. Compared with SD, the exposure to MF resulted in robust transcript and miR expression changes in terms of the number and magnitude. For data analysis, statistically significant mRNAs (2-fold) and miRs (1.5-fold) were processed by Ingenuity Pathway Analysis to uncover miRs associated with target transcripts from several cellular pathways after irradiation. Interestingly, MF radiation induced a cohort of mRNAs and miRs that coordinate the induction of immune response pathway under tight regulation. In addition, mRNAs and miRs associated with DNA replication, recombination and repair, apoptosis, cardiovascular events, and angiogenesis were revealed.

Implications: Radiation-induced alterations in stress and immune response genes in endothelial cells contribute to changes in normal tissue and tumor microenvironment, and affect the outcome of radiotherapy. Mol Cancer Res; 12(7); 1002–15. ©2014 AACR.


Hypoxia induces genomic instability through replication stress and dysregulation of vital DNA repair pathways. The Fanconi anemia (FA) proteins, FANCD2 and FANCI, are key members of a DNA repair pathway that responds to replicative stress, suggesting that they undergo regulation by hypoxic conditions. Here acute hypoxic stress activates the FA pathway via ubiquitination of FANCD2 and FANCI in an ATR-dependent manner. In addition, the presence of an intact FA pathway is required for preventing hypoxia-induced DNA damage measurable by the comet assay, limiting the accumulation of H2AX (a marker of DNA damage or stalled replication), and protecting cells from hypoxia-induced apoptosis. Furthermore, prolonged hypoxia induces transcriptional repression of FANCD2 in a manner analogous to the hypoxic downregulation of BRCA1 and RAD51. Thus, hypoxia-induced FA pathway activation plays a key role in maintaining genome integrity and cell survival, while FA protein downregulation with prolonged hypoxia contributes to genomic instability.

Implications: This work highlights the critical role of the FA pathway in response to hypoxic stress and identifies the pathway as a therapeutic target under hypoxic conditions. Mol Cancer Res; 12(7); 1016–28. ©2014 AACR.


African Americans are disproportionately affected by early-onset, high-grade malignancies. A fraction of this cancer health disparity can be explained by genetic differences between individuals of African or European descent. Here the wild-type Pro/Pro genotype at the TP53Pro72Arg (P72R) polymorphism (SNP: rs1042522) is more frequent in African Americans with cancer than in African Americans without cancer (51% vs. 37%), and is associated with a significant increase in the rates of cancer diagnosis in African Americans. To test the hypothesis that Tp53 allele–specific gene expression may contribute to African American cancer disparities, TP53 hemizygous knockout variants were generated and characterized in the RKO colon carcinoma cell line, which is wild type for TP53 and heterozygous at the TP53Pro72Arg locus. Transcriptome profiling, using RNAseq, in response to the DNA-damaging agent etoposide revealed a large number of Tp53-regulated transcripts, but also a subset of transcripts that were TP53Pro72Arg allele specific. In addition, a shRNA-library suppressor screen for Tp53 allele–specific escape from Tp53-induced arrest was performed. Several novel RNAi suppressors of Tp53 were identified, one of which, PRDM1β (BLIMP-1), was confirmed to be an Arg-specific transcript. Prdm1β silences target genes by recruiting H3K9 trimethyl (H3K9me3) repressive chromatin marks, and is necessary for stem cell differentiation. These results reveal a novel model for African American cancer disparity, in which the TP53 codon 72 allele influences lifetime cancer risk by driving damaged cells to differentiation through an epigenetic mechanism involving gene silencing.

Implications: TP53 P72R polymorphism significantly contributes to increased African American cancer disparity. Mol Cancer Res; 12(7); 1029–41. ©2014 AACR.


Activating mutations and/or overexpression of FGFR3 are common in bladder cancer, making FGFR3 an attractive therapeutic target in this disease. In addition, FGFR3 gene rearrangements have recently been described that define a unique subset of bladder tumors. Here, a selective HSP90 inhibitor, ganetespib, induced loss of FGFR3-TACC3 fusion protein expression and depletion of multiple oncogenic signaling proteins in RT112 bladder cells, resulting in potent cytotoxicity comparable with the pan-FGFR tyrosine kinase inhibitor BGJ398. However, in contrast to BGJ398, ganetespib exerted pleiotropic effects on additional mitogenic and survival pathways and could overcome the FGFR inhibitor–resistant phenotype of FGFR3 mutant–expressing 97-7 and MHG-U3 cells. Combinatorial benefit was observed when ganetespib was used with BGJ398 both in vitro and in vivo. Interestingly, two additional FGFR3 fusion-positive lines (RT4 and SW480) retained sensitivity to HSP90 inhibitor treatment by the ansamycins 17-AAG and 17-DMAG yet displayed intrinsic resistance to ganetespib or AUY922, both second-generation resorcinol-based compounds. Both cell lines, compared with RT112, expressed considerably higher levels of endogenous UGT1A enzyme; this phenotype resulted in a rapid glucuronidation-dependent metabolism and subsequent efflux of ganetespib from SW780 cells, thus providing a mechanism to account for the lack of bioactivity.

Implications: Pharmacologic blockade of the molecular chaperone HSP90 represents a promising approach for treating bladder tumors driven by oncogenic gene rearrangements of FGFR3. Furthermore, UDP-glucuronosyltransferase enzyme expression may serve as a predictive factor for clinical response to resorcinol-based HSP90 inhibitors. Mol Cancer Res; 12(7); 1042–54. ©2014 AACR.


TBK1 (TANK-binding kinase 1) is a noncanonical IB protein kinase that phosphorylates and activates downstream targets such as IRF3 and c-Rel and, mediates NF-B activation in cancer. Previous reports demonstrated synthetic lethality of TBK1 with mutant KRAS in non–small cell lung cancer (NSCLC); thus, TBK1 could be a novel target for treatment of KRAS-mutant NSCLC. Here, the effect of TBK1 on proliferation in a panel of cancer cells by both genetic and pharmacologic approaches was evaluated. In KRAS-mutant cancer cells, reduction of TBK1 activity by knockdown or treatment with TBK1 inhibitors did not correlate with reduced proliferation in a two-dimensional viability assay. Verification of target engagement via reduced phosphorylation of S386 of IRF3 (pIRF3S386) was difficult to assess in NSCLC cells due to low protein expression. However, several cell lines were identified with high pIRF3S386 levels after screening a large panel of cell lines, many of which also harbor KRAS mutations. Specifically, a large subset of KRAS-mutant pancreatic cancer cell lines was uncovered with high constitutive pIRF3S386 levels, which correlated with high levels of phosphorylated S172 of TBK1 (pTBK1S172). Finally, TBK1 inhibitors dose-dependently inhibited pIRF3S386 in these cell lines, but this did not correlate with inhibition of cell growth. Taken together, these data demonstrate that the regulation of pathways important for cell proliferation in some NSCLC, pancreatic, and colorectal cell lines is not solely dependent on TBK1 activity.

Implications: TBK1 has therapeutic potential under certain contexts and phosphorylation of its downstream target IRF3 is a biomarker of TBK1 activity. Mol Cancer Res; 12(7); 1055–66. ©2014 AACR.