As genomics advances reveal the cancer gene landscape, a daunting task is to understand how these genes contribute to dysregulated oncogenic pathways. Integration of cancer genes into networks offers opportunities to reveal protein-protein interactions (PPIs) with functional and therapeutic significance. Here, we report the generation of a cancer-focused PPI network, termed OncoPPi, and identification of >260 cancer-associated PPIs not in other large-scale interactomes. PPI hubs reveal new regulatory mechanisms for cancer genes like MYC, STK11, RASSF1 and CDK4. As example, the NSD3 (WHSC1L1)-MYC interaction suggests a new mechanism for NSD3/BRD4 chromatin complex regulation of MYC-driven tumours. Association of undruggable tumour suppressors with drug targets informs therapeutic options. Based on OncoPPi-derived STK11-CDK4 connectivity, we observe enhanced sensitivity of STK11-silenced lung cancer cells to the FDA-approved CDK4 inhibitor palbociclib. OncoPPi is a focused PPI resource that links cancer genes into a signalling network for discovery of PPI targets and network-implicated tumour vulnerabilities for therapeutic interrogation.
The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kDa and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3ζ in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer.
API-1 is a novel small molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation, and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of c-FLIP levels and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of DR4 or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis, but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1, but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Since other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity.
Background
Lonafarnib (LNF) is a protein farnesyl transferase (FTase) inhibitor that has shown synergistic activity with taxanes in preclinical models and early stage clinical trials. Preclinical findings suggested tubulin acetylation and FTase expression levels may be important determinants of drug sensitivity that would help identify patient populations more likely to benefit from this regimen. This pilot study evaluated the biological effects of LNF and docetaxel (DTX) combination therapy in refractory solid tumors by comparing pre- and post-treatment tumor biopsies.
Methods
Patients with histologically-confirmed locally advanced or metastatic solid malignancies refractory to standard therapies or with no effective therapies available were eligible. Patients were randomized to one of four dosing cohorts: (1) 30mg/m2, 100mg, (2) 36mg/m2, 100mg, (3) 30mg/m2, 150mg, or (4) 36mg/m2, 150mg of DTX IV weekly, LNF PO BID, respectively.
Results
Of the 38 patients enrolled, 36 were treated, and 29 were evaluable for toxicity and response assessment. The combination of LNF and DTX was tolerated in all cohorts with the exception of a 28% incidence of grade 3/4 diarrhea which was manageable with aggressive anti-diarrheal regimens. Seven patients derived clinically meaningful benefit from this combination treatment; these patients had significantly lower basal FTase-beta mRNA expression levels than the mean study population level (p<0.05). Correlation of clinical benefit with tubulin acetylation content as well as basal acetyl-tubulin content were evaluated. However, no significant correlation was found.
Conclusions
Despite the small number of patients, these findings support our preclinical mechanistic studies and warrant further clinical investigations using FTase-beta mRNA expression as a potential predictive biomarker to select for an enriched patient population to study the effects of taxane and FTase inhibitor combination therapies.
EAPII (also called TTRAP, TDP2), a protein identified a decade ago, has recently been shown to function as an oncogenic factor. This protein was also proven to be the first 5′-tyrosyl-DNA phosphodiesterase. EAPII has been demonstrated to have promiscuous protein associations, broad responsiveness to various extracellular signals and pleiotropic functions in the development of human diseases including cancer and neurodegenerative disease. Emerging data suggest that EAPII is a multi-functional protein: EAPII repairs enzyme (topoisomerase)-mediated DNA damage by removing phosphotyrosine from DNA adducts; EAPII is involved in multiple signal transduction pathways such as TNF-TNFR, TGF? and MAPK, and EAPII is responsive to immune defense, inflammatory response, virus infection and DNA toxins (chemo or radiation therapy). This review focuses on the current understanding of EAPII biology and its potential relations to many aspects of cancer development, including chromosome instability, tumorigenesis, tumor metastasis and chemoresistance, suggesting it as a potential target for intervention in cancer and other human diseases.
Cigarette smoking, either active or passive, is the most important risk factor in the development of human lung cancer. Mounting evidence indicates that cigarette smoke constituents not only contribute to tumorigenesis but also may increase the spread of cancer in the body. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen. NNK, an important component in cigarette smoke, may also promote tumor metastasis by regulating cell motility. Here we found that NNK can induce activation of a functionally interdependent protein kinase cascade, including c-Src, PKCι and FAK, in association with increased migration and invasion of human lung cancer cells. c-Src, PKCι and FAK are extensively co-localized in the cytoplasm. Treatment of cells with α7 nAChR specific inhibitor α-bungarotoxin (α-BTX) blocks NNK-stimulated activation of c-Src, PKCι and FAK and suppresses cell migration and invasion. Intriguingly, NNK enhances c-Src/PKCι and PKCι/FAK bindings, indicating a potential mechanism by which these kinases activate each other. Specific disruption of c-Src, PKCι or FAK expression by RNA interference significantly reduces NNK-induced cell migration and invasion. These findings suggest that NNK-induced migration and invasion may occur in a mechanism through activation of a c-Src/PKCι/FAK loop, which can contribute to metastasis and/or development of human lung cancer.
Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate, and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.
Inhibition of mTOR signaling by rapamycin has been demonstrated to activate ERK1/2 and Akt in various types of cancer cells, which contributes to rapamycin resistance. However, the downstream effect of rapamycin-activated ERKs and Akt on survival or death substrate(s) remains unclear. We discovered that treatment of human lung cancer cells with rapamycin results in enhanced phosphorylation of Bad at serine (S) 112 and S136 but not S155 in association with activation of ERK1/2 and Akt. A higher level of Bad phosphorylation was observed in rapamycin-resistant cells compared to parental rapamycin-sensitive cells. Thus, Bad phosphorylation may contribute to rapamycin resistance. Mechanistically, rapamycin promotes Bad accumulation in the cytosol, enhances Bad/14-3-3 interaction and reduces Bad/Bcl-XL binding. Rapamycin-induced Bad phosphorylation promotes its ubiquitination and degradation, with a significant reduction of its half-life (i.e. from 53.3 h to 37.5 h). Inhibition of MEK/ERK by PD98059 or depletion of Akt by RNA interference blocks rapamycin-induced Bad phosphorylation at S112 or S136, respectively. Simultaneous blockage of S112 and S136 phosphorylation of Bad by PD98059 and silencing of Akt significantly enhances rapamycin-induced growth inhibition in vitro and synergistically increases the anti-tumor efficacy of rapamycin in lung cancer xenografts. Intriguingly, either suppression of Bad phosphorylation at S112 and S136 sites or expression of the non-phosphorylatable Bad mutant (S112A/S136A) can reverse rapamycin resistance. These findings uncover a novel mechanism of rapamycin resistance, which may promote the development of new strategies for overcoming rapamycin resistance by manipulating Bad phosphorylation at S112 and S136 in human lung cancer.
Bax, a central death regulator, is required at the decisional stage of apoptosis. We recently identified serine 184 (S184) of Bax as a critical functional switch controlling its proapoptotic activity. Here we used the structural pocket around S184 as a docking site to screen the NCI library of small molecules using the UCSF-DOCK programme suite. Three compounds, small-molecule Bax agonists SMBA1, SMBA2 and SMBA3, induce conformational changes in Bax by blocking S184 phosphorylation, facilitating Bax insertion into mitochondrial membranes and forming Bax oligomers. The latter leads to cytochrome c release and apoptosis in human lung cancer cells, which occurs in a Bax- but not Bak-dependent fashion. SMBA1 potently suppresses lung tumour growth via apoptosis by selectively activating Bax in vivo without significant normal tissue toxicity. Development of Bax agonists as a new class of anticancer drugs offers a strategy for the treatment of lung cancer and other Bax-expressing malignancies.
Inhibition of BET bromodomains (BRDs) has emerged as a promising cancer therapeutic strategy. Accordingly, inhibitors of BRDs such as JQ1 have been actively developed and some have reached clinical testing. However, the mechanisms by which this group of inhibitors exerts their anticancer activity, including induction of apoptosis, have not been fully elucidated. This report reveals a previously uncovered activity of JQ1 in inducing c-FLIP degradation and enhancing TRAIL-induced apoptosis. JQ1 potently decreased c-FLIP (both long and short forms) levels in multiple cancer cell lines without apparently increasing the expression of DR5 and DR4. Consequently, JQ1, when combined with TRAIL, synergistically induced apoptosis; this enhanced apoptosis-inducing activity could be abolished by enforced expression of ectopic FLIPL or FLIPS. Hence it appears that JQ1 decreases c-FLIP levels, resulting in enhancement of TRAIL-induced apoptosis. Inhibition of proteasome with MG132 prevented JQ1-induced c-FLIP reduction. Moreover, JQ1 decreased c-FLIP stability. Therefore, JQ1 apparently decreases c-FLIP levels through facilitating its proteasomal degradation. Genetic inhibition of either BRD4 or c-Myc by knocking down their expression failed to mimic JQ1 in decreasing c-FLIP and enhancing TRAIL-induced apoptosis, suggesting that JQ1 induces c-FLIP degradation and enhances TRAIL-induced apoptosis independent of BRD4 or c-Myc inhibition. In summary, our findings in this study highlights a novel biological function of JQ1 in modulating apoptosis and warrant further study of the potential treatment of cancer with the JQ1 and TRAIL combination.