Small ubiquitin-like modifier (SUMO) family proteins regulate target-protein functions by post-translational modification. However, a potent and selective inhibitor targeting the SUMO pathway has been lacking. Here we describe ML-792, a mechanism-based SUMO-activating enzyme (SAE) inhibitor with nanomolar potency in cellular assays. ML-792 selectively blocks SAE enzyme activity and total SUMOylation, thus decreasing cancer cell proliferation. Moreover, we found that induction of the MYC oncogene increased the ML-792-mediated viability effect in cancer cells, thus indicating a potential application of SAE inhibitors in treating MYC-amplified tumors. Using ML-792, we further explored the critical roles of SUMOylation in mitotic progression and chromosome segregation. Furthermore, expression of an SAE catalytic-subunit (UBA2) S95N M97T mutant rescued SUMOylation loss and the mitotic defect induced by ML-792, thus confirming the selectivity of ML-792. As a potent and selective SAE inhibitor, ML-792 provides rapid loss of endogenously SUMOylated proteins, thereby facilitating novel insights into SUMO biology.
Abstract. Predicting tumor metastatic potential remains a challenge in cancer research and clinical practice. Our goal was to identify novel biomarkers for differentiating human breast tumors with different metastatic potentials by imaging the in vivo mitochondrial redox states of tumor tissues. The more metastatic ͑aggressive͒ MDA-MB-231 and less metastatic ͑indolent͒ MCF-7 human breast cancer mouse xenografts were imaged with the low-temperature redox scanner to obtain multi-slice fluorescence images of reduced nicotinamide adenine dinucleotide ͑NADH͒ and oxidized flavoproteins ͑Fp͒. The nominal concentrations of NADH and Fp in tissue were measured using reference standards and used to calculate the Fp redox ratio, Fp/ ͑NADH+ Fp͒. We observed significant core-rim differences, with the core being more oxidized than the rim in all aggressive tumors but not in the indolent tumors. These results are consistent with our previous observations on human melanoma mouse xenografts, indicating that mitochondrial redox imaging potentially provides sensitive markers for distinguishing aggressive from indolent breast tumor xenografts. Mitochondrial redox imaging can be clinically implemented utilizing cryogenic biopsy specimens and is useful for drug development and for clinical diagnosis of breast cancer.
Noninvasive or minimally invasive prediction of tumor metastatic potential would facilitate individualized cancer management. Studies were performed on a panel of human melanoma xenografts that spanned the full range of metastatic potential measured by an in vivo lung colony assay and an in vitro membrane invasion culture system. Three imaging methods potentially transferable to the clinic [dynamic contrast-enhanced (DCE) MRI, T1-MRI, and low-temperature fluorescence imaging (measurable on biopsy specimens)] distinguished between relatively less metastatic and more metastatic human melanoma xenografts in nude mice. DCE-MRI, analyzed with the shutterspeed relaxometric algorithm and using an arterial input function simultaneously measured in the left ventricle of the mouse heart, yielded a blood transfer rate constant, Ktrans, that measures vascular perfusion/permeability. Ktrans was significantly higher in the core of the least metastatic melanoma (A375P) than in the core of the most metastatic melanoma (C8161). C8161 melanoma had more blood vascular structures but fewer functional blood vessels than A375P melanoma. The A375P melanoma exhibited mean T1 values that were significantly higher than those of C8161 melanoma. Measurements of T1 and T2 relaxation times did not differ significantly between these 2 melanomas. The mitochondrial redox ratio, Fp/(Fp ؉ NADH), where Fp and NADH are the fluorescences of oxidized flavoproteins and reduced pyridine nucleotides, respectively, varied linearly with the in vitro invasive potential of the 5 melanoma cell lines (A375P, A375M, A375P10, A375P5, and C8161). This study shows that a harsh microenvironment may promote melanoma metastasis and provides potential biomarkers of metastatic potential. dynamic contrast enhanced MRI ͉ mitochondrial redox state ͉ T1rho ͉ invasive potential ͉ human melanoma xenografts M elanoma is treated primarily by surgical excision, which is often curative if the tumor is detected in its early stages. However, if recurrence with metastasis occurs, the prognosis is very poor because effective methods for treating systemic disease are not available. Evaluation of the metastatic potential of a melanoma at the time of surgery could determine the aggressiveness of the surgical procedures to be undertaken and the frequency of postsurgical surveillance.Criteria currently available for staging human melanoma malignancies and predicting their metastatic potential include histopathological evaluation, height of the lesion, disease progression to sentinel lymph nodes, and genomic and proteomic approaches currently under development and evaluation (1-5). The objective of this study was to explore a variety of noninvasive and biopsy-based imaging methods that could be used to better distinguish between aggressive and indolent neoplasms and to identify biomarkers of tumor aggressiveness.We chose to study melanoma because of the availability of a panel of human melanoma cell lines and their corresponding xenografts in immunosuppressed mice that span the full rang...
SUMOylation is a reversible post-translational modification that regulates protein function through covalent attachment of small ubiquitin-like modifier (SUMO) proteins. The process of SUMOylating proteins involves an enzymatic cascade, the first step of which entails the activation of a SUMO protein through an ATP-dependent process catalyzed by SUMO-activating enzyme (SAE). Here, we describe the identification of TAK-981, a mechanism-based inhibitor of SAE which forms a SUMO−TAK-981 adduct as the inhibitory species within the enzyme catalytic site. Optimization of selectivity against related enzymes as well as enhancement of mean residence time of the adduct were critical to the identification of compounds with potent cellular pathway inhibition and ultimately a prolonged pharmacodynamic effect and efficacy in preclinical tumor models, culminating in the identification of the clinical molecule TAK-981.
An effective method for in vivo detection of early therapeutic response of patients with non-Hodgkin's lymphoma would enable personalized clinical management of cancer therapy and facilitate the design of optimal treatment regimens. This study evaluates the feasibility of T 2 -weighted MRI (T2WI) and diffusion-weighted MRI (DWI) for in vivo detection of response of human diffuse large B-cell lymphoma xenografts in severe combined immunodeficient mice to chemotherapy. Each cycle of combination chemotherapy with cyclophosphamide, hydroxydoxorubicin, Oncovin, prednisone, and bryostatin 1 (CHOPB) was administered to tumor-carrying mice weekly for up to four cycles. T2WI and DWI were performed before the initiation of CHOPB and after each cycle of CHOPB. In order to corroborate the MRI results, histological analyses were carried out on control tumors and treated tumors after completion of all MRI studies. DWI revealed a significant (P < 0.03) increase in the mean apparent diffusion coefficient in CHOPB-treated tumors as early as 1 week after initiation of CHOPB. However, a significant (P < 0.03) decrease in mean T 2 was observed only after two cycles of CHOPB. Both MRI methods produced high-resolution (0.1 Â 0.1 Â 1.0 mm 3 ) maps of regional therapeutic response in the treated tumors based on local apparent diffusion coefficient and T 2 . Only a specific region of the tumors (in 3 of the 5 tumors) corresponding to about one third of the tumor volume exhibited a response-associate increase in ADC and decrease in T 2 . An adjacent region exhibited an increase in T 2 and no change in ADC. The rest of the tumor was indistinguishable from shamtreated controls by MRI criteria. The therapeutic response of the treated tumors detected by MRI was accompanied by changes in tumor cell density, proliferation and apoptosis revealed by histological studies performed upon completion of the longitudinal study. The mechanism producing the regional response of the tumor remains to be elucidated.
Rationale and Objectives Cancer cells generate more lactate than normal cells under aerobic and hypoxic conditions – exhibiting the so-called Warburg effect. However, the relationship between the Warburg effect and tumor metastatic potential remains controversial. We intend to investigate whether the higher lactate reflects higher tumor metastatic potential. Materials and Methods We employed hyperpolarized 13C-pyruvate magnetic resonance spectroscopy (MRS) to compare lactate 13C-labeling in vivo in breast cancer mouse xenografts of highly metastatic (MDA-MB-231) and relatively indolent (MCF-7) human cell lines. We obtained the kinetic parameters of the lactate dehydrogenase (LDH)-catalyzed reaction by three methods of analysis including the differential equation (DE) fit, q-ratio (qR) fit, and ratio fit (RF) methods. Results Consistent results from the three methods showed that the highly metastatic breast tumors exhibited a smaller apparent forward rate constant (k+ = 0.060 ± .004 s−1) than the relatively indolent tumors (k+ = 0.097 ± .013 s−1). The RF fit generates the highest statistical significance for the difference (p=0.02). No significant difference is found for the reverse rate constant. Conclusion The result indicates that the less metastatic breast tumors may produce more lactate than the highly metastatic ones from the injected 13C-pyruvate, and supports the viewpoint that breast tumor metastatic risk is not necessarily associated with the high levels of glycolysis and lactate production. More studies are needed to confirm whether and how much the measured apparent rate constants are affected by the membrane transporter activity and whether they are primarily determined by the LDH activity or not.
Purpose Tissue redox state is an important mediator of various biological processes in health and diseases such as cancer. Previously, we discovered that the mitochondrial redox state of ex vivo tissues detected by redox scanning (an optical imaging method) revealed interesting tumor redox state heterogeneity that could differentiate tumor aggressiveness. Because the noninvasive chemical exchange saturation transfer (CEST) MRI can probe the proton transfer and generate contrasts from endogenous metabolites, we aim to investigate if the in vivo CEST contrast is sensitive to proton transfer of the redox reactions so as to reveal the tissue redox states in breast cancer animal models. Procedures CEST MRI has been employed to characterize tumor metabolic heterogeneity and correlated with the redox states measured by the redox scanning in two human breast cancer mouse xenograft models, MDA-MB-231 and MCF-7. The possible biological mechanism on the correlation between the two imaging modalities was further investigated by phantom studies where the reductants and the oxidants of the representative redox reactions were measured. Results The CEST contrast is found linearly correlated with NADH concentration and the NADH redox ratio with high statistical significance, where NADH is the reduced form of nicotinamide adenine dinucleotide. The phantom studies showed that the reductants of the redox reactions have more CEST contrast than the corresponding oxidants, indicating that higher CEST effect corresponds to the more reduced redox state. Conclusions This preliminary study suggests that CEST MRI, once calibrated, might provide a novel noninvasive imaging surrogate for the tissue redox state and a possible diagnostic biomarker for breast cancer in the clinic.
The invasive/metastatic potential of cancer cells is an important factor in tumor progression. The redox ratios obtained from ratios of the endogenous fluorescent signals of NADH and FAD, can effectively respond to the alteration of cancer cells in its mitochondrial energy metabolism. It has been shown previously that the redox ratios may predict the metastatic potential of cancer mouse xenografts. In this report, we aimed to investigate the metabolic state represented by the redox ratios of cancer cells in vitro. Fluorescence microscopic imaging technology was used to observe the changes of the endogenous fluorescence signals of NADH and FAD in the energy metabolism pathways. We measured the redox ratios (FAD/NADH) of breast cancer cell lines MDA-MB-231, MDA-MB-468, MCF-7, and SKBR3. We found that the more invasive cancer cells have higher FAD/NADH ratios, largely consistent with previous studies on breast cancer xenografts. Furthermore, by comparing the fluorescence signals of the breast cancer cells under different nutritional environments, including starvation and addition of glutamine, pyruvate and lactate, we found that the redox ratios still effectively distinguished the highly invasive MDA-MB-231 cells from less invasive MCF-7 cells. These preliminary data suggest that the redox ratio may potentially provide a new index to stratefy breast cancer with different degrees of aggressiveness, which could have significance for the diagnosis and treatment of breast cancer.
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