Mir-17-5p Regulates Breast Cancer Cell Proliferation by Inhibiting Translation of AIB1 mRNA

MicroRNAs are an extensive family of ∼22-nucleotide-long noncoding RNAs expressed in a wide range of eukaryotes, including humans, and they are important in development and disease. We found that microRNA Mir-17-5p has extensive complementarity to the mRNA of AIB1 (named for “amplified in breast cancer 1”). Cell culture experiments showed that AIB1 expression was downregulated by Mir-17-5p, primarily through translational inhibition. Expression of Mir-17-5p was low in breast cancer cell lines. We also found that downregulation of AIB1 by Mir-17-5p resulted in decreased estrogen receptor-mediated, as well as estrogen receptor-independent, gene expression and decreased proliferation of breast cancer cells. Mir-17-5p also completely abrogated the insulin-like growth factor 1-mediated, anchorage-independent growth of breast cancer cells. Our results reveal that Mir-17-5p has a role as a tumor suppressor in breast cancer cells

Abstract 4096: TRAIL enhances cytotoxicity of arginine depletion therapy in argininosuccinate synthetase-negative melanoma cells through interruption of autophagy via activation of caspases

Abstract Melanomas which do not express argininosuccinate synthetase (ASS) cannot make arginine from citrulline; thus arginine depletion caused by pegylated arginine deiminase (ADI-PEG20, provided by Polaris Inc.) results in growth inhibition and eventually leads to cell death. However, autophagy does take place during arginine deprivation which prevents cell death. During this period, certain melanoma cells can turn on the ASS gene and hence become resistant to this form of treatment. In order to increase the effectiveness of ADI-PEG20 therapy, we have previously found that combining tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with ADI-PEG20 can accelerate cell death (apoptosis) process and hence greatly enhance cytotoxicity (Biochem. Biophys. Res. Commun. 2010; 394(3):760-6). We have found that the addition of TRAIL increases formation of tBid. In this study, we found that with the combination treatment, autophagy-related protein Atg5 and Beclin-1 are cleaved which suggests that autophagic process is interrupted and apoptosis can take place. In order to investigate whether caspase activation plays a role in Atg5 and Beclin-1 cleavage, we have used specific caspase inhibitors (all at 25 nM) to address this question. A375 (ASS non-inducible) and A2058 (ASS inducible) cell lines were used for this study. About 70-80 % cell death occurs at 24 h after combination treatment, while only 10-20% cell death occurs when treated with TRAIL alone. Almost no cell death is apparent when treated with arginine-free medium alone at this time point. Adding caspase-8 (Z-IETD-FMK), caspase-9 (Z-LEHD-FMK) or caspase-10 (Z-AEVD-FMK) single inhibitor can almost completely rescue cell death on combination treatment while caspase-6 inhibitor (Z-VEID-FMK) has less rescuing effect (about 10-30% cell death). Inhibitors for caspase-3 (Z-DEVD-FMK) or 3/7 (Ac-DNLD-CHO) have least effect (more than 50% cell death). These data confirm that the enhancement effect of the combination needs the participation of both the extrinsic and intrinsic pathways of apoptosis. The cleavage of Beclin-1 and Atg5 are also least affected when caspase-3 or 3/7 inhibitor is applied, but blocked or reduced when single inhibitor to caspase-6, 8, 10 or 9 is added. There is little difference between the ASS inducible or non-inducible cell lines. This finding suggests that the combination treatment turns off the autophagic process during arginine deprivation, possibly through caspase activation and allows apoptotic process to take place. Overall, our findings indicate that autophagy is a pro-survival mechanism in melanoma cells under nutritional stress, and interfering with this pathway may achieve greater efficacy with ADI-PEG20 treatment. (Supported by NIH grant 1R01CA109578) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4096. doi:10.1158/1538-7445.AM2011-4096</jats:p

Abstract 4404: Novel approach to circumvent cisplatin resistant lung cancer by targeting metabolism

Abstract For the past three decades, there are no drugs which could reverse cisplatin resistance or selectively kill these resistant cells. We have discovered that cisplatin resistant (CR) lung cancer shares one common factor which is increase in reactive oxygen species (ROS). Decreased intracellular thioredoxin-1(TRX1) due to excessive secretion was found in vitro and in vivo, as well as in patients’ serum which could be a primary contributory factor to higher mitochondria-ROS levels. Furthermore, CR cells possess significantly increased number of mitochondria and consume higher rates of oxygen. Key glycolytic enzymes (HKII and LDHA) and lactate production were decreased in CR cells which suggests rewiring of tumor metabolic pathways. NAD+, crucial co-factor of all redox systems, is significantly reduced in all CR cells. Importantly, treatment with riluzole (the FDA approved drug that interferes with cystine/glutamate pump and results in reduced intracellular glutathione (GSH) levels) led to further decrease in NAD+ and LDHA, as well as heightened oxidative stress in CR cells. ROS levels were increased (2-3X) more in resistant cells after riluzole treatment while no significant change occurred in parental cells. The ID50 dosage of parental cells were 3-4 fold more than theirs CR cell counterparts and no cytotoxicity were found in normal lung fibroblast (n=8; p&amp;lt;0.05). Addition of 200 µM NAD+ re-stabilized LDHA, reduced ROS, and reversed riluzole induced cell death. To further determine whether LDHA correlates with ROS levels and sensitivity to riluzole, we inhibited LDHA expressions in parental cell lines S and H460 using siRNA. Lactate productions were decreased in knockdown cells and further attenuated upon riluzole treatment when compared with scrambled control. Higher basal levels of ROS were obtained from S+siLDHA and H460+siLDHA and further increased in ROS levels were observed when treated with riluzole. We then evaluated the viability of these cells under riluzole treatment. Both siLDHA clones were sensitive to riluzole particularly in S+siLDHA which showed 4 fold more sensitivity. Importantly, treatment of riluzole also led to further decrease in LDHA expressions at both protein and mRNA levels. Using riluzole in 2 mouse xenograft models (H460CR (KRAS mutant) and SC (wt)) resulted in completely disappeared in wild type mice and were significant reduced in KRAS mutant mice (n=5 per treatment group; p&amp;lt;0.002). Repurpose of riluzole should be considered for future treatment of CR lung cancer patients. Supported by Department of Veterans Affairs, CDA2 award (1K2BX001289) and Woman Cancer Association Fund. Citation Format: Medhi Wangpaichitr, Chunjing Wu, Ying Ying Li, Lynn G. Feun, Macus T. Kuo, Niramol Savaraj. Novel approach to circumvent cisplatin resistant lung cancer by targeting metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4404. doi:10.1158/1538-7445.AM2017-4404</jats:p

Abstract 1692: Downregulation of thioredoxin-1 confers resistance to cisplatin and sensitivity to the ROS generating agent elesclomol

Abstract We have previously discovered a unique and important finding that all of our cisplatin resistant (CR) small cell and non small cell lung cancer cells (5 pairs of cell lines, parental vs. CR cells, and one primary line tested) possess high levels of ROS (Reactive Oxygen Species) compared to their parental cancer cell counterparts and normal cells. Importantly, these CR cells are sensitive to elesclomol (Synta Pharmaceuticals), a new compound which kills cells by generating ROS (Cancers, 23-28; 2009). The question remains why these CR cells possess intrinsically high ROS levels. We have found that the common biochemical feature in CR cells is lower expression of the antioxidant protein thioredoxin-1 (TRX1). CR cells have an average of 6-8 fold lower TRX1 as compared to their parental counterparts and normal cells. Other antioxidant systems either showed no changes or elevated such as GSH but varies among cell lines. To further verify that TRX1 is an important contributory factor to the high ROS levels been in CR cells, we knocked down TRX1 in parental cells (SCLC1) using siRNA. These SCLC1/TRX1(−) transfectants generated significantly higher ROS (P&amp;lt;0.001) when compared to the control. SCLC1/TRX1(−) cells are resistant to cisplatin with the ID50 of 1.1 µg/ml vs. 0.2 µg/ml in the control transfectants. Moreover, SCLC1/TRX1(−) cells are also hypersensitive to elesclomol (ID50 of 18nM vs. 45nM). Correspondingly, overexpressing TRX1 in the CR cell line SR2, using the pCMV6 vector which contains the full length cDNA of TRX1, resulted in decreased ROS production but increased sensitivity to cisplatin. The ID50 of cisplatin in SR2/TRX1(+) transfectant was 1.2 µg/ml compared to 2.6 µg/ml in SR2 cells. SR2/TRX1(+) cells also became more resistant to elesclomol (ID50 of 35nM vs. 6nM). Thus, our data strongly indicate that TRX1 is a major contributory factor in higher ROS levels found in CR lung cancer cells. Interestingly, it has been reported that diminished elesclomol activity is influenced by high LDHA levels. Our data show that all CR cells have 3-5 fold lower levels of LDHA levels when compared with their parental cells. Importantly, SR2/TRX1(+) also exhibits higher LDHA levels. Thus, it is possible that ROS production seen in CR cells may be in part due to low TRX1 levels resulting in altered cellular metabolism (more OXPHOS) and lower LDHA. Treatment of these cells with elesclomol results in a greater increase in ROS levels beyond their tolerance leading to cell death. Lung tumor cells which have higher LDHA most likely have lower basal ROS levels and retain the ability to turn on TRX1 system. Thus, ROS generation by elesclomol is unable to eradicate these tumor cells. Our findings suggest another novel approach to selectively kill CR lung tumors which intrinsically produce higher ROS and express lower TRX1 and LDHA levels. Supported by VA Merit Review. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1692. doi:10.1158/1538-7445.AM2011-1692</jats:p

Enhancing the Effect of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Signaling and Arginine Deprivation in Melanoma

Melanoma as a very aggressive type of cancer is still in urgent need of improved treatment. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and arginine deiminase (ADI-PEG20) are two of many suggested drugs for treating melanoma. Both have shown anti-tumor activities without harming normal cells. However, resistance to both drugs has also been noted. Studies on the mechanism of action of and resistance to these drugs provide multiple targets that can be utilized to increase the efficacy and overcome the resistance. As a result, combination strategies have been proposed for these drug candidates with various other agents, and achieved enhanced or synergistic anti-tumor effect. The combination of TRAIL and ADI-PEG20 as one example can greatly enhance the cytotoxicity to melanoma cells including those resistant to the single component of this combination. It is found that combination treatment generally can alter the expression of the components of cell signaling in melanoma cells to favor cell death. In this paper, the signaling of TRAIL and ADI-PEG20-induced arginine deprivation including the main mechanism of resistance to these drugs and exemplary combination strategies is discussed. Finally, factors hampering the clinical application of both drugs, current and future development to overcome these hurdles are briefly discussed

TRAIL induces autophagic protein cleavage through caspase activation in melanoma cell lines under arginine deprivation

Arginine deprivation is a promising strategy for treating ASS-negative malignant tumors including melanoma. However, autophagy can potentially counteract the effectiveness of this treatment by acting as a pro-survival pathway. By combining TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) with arginine deprivation using ADI-PEG20 (pegylated arginine deiminase), we achieved enhanced apoptosis and accelerated cell death in melanoma cell lines. This implies a switch from autophagy to apoptosis. In our current investigation, we found that TRAIL could induce the cleavage of two key autophagic proteins, Beclin-1 and Atg5, in the combination treatment. Using specific inhibitors for individual caspases, we found caspase-8 inhibitor could completely abolish the cleavage. Furthermore, caspase-8 inhibitor was able to fully reverse the enhanced cytotoxicity induced by TRAIL. Inhibitors for caspase-3, 6, 9, and 10 were able to block the cleavage of these two autophagic proteins to some extent and correspondingly rescue cells from the cytotoxicity of the combination of TRAIL and arginine deprivation. In contrast, calpain inhibitor could not prevent the cleavage of either Beclin-1 or Atg5, and was unable to prevent cell death. Overall, our data indicate that the cleavage of Beclin-1 and Atg5 by TRAIL-initiated caspase activation is one of the mechanisms that lead to the enhancement of the cytotoxicity in the combination treatment

Abstract 1138: The emerging role of thioredoxin-1 on ROS and metabolic reprogramming in cisplatin resistant lung cancer

Abstract Cisplatin resistance remains a major problem in treating lung cancer. We have discovered that all cisplatin resistant (CR) cells, regardless of their signaling mechanism, possess decreased levels of thioredoxin-1 (TRX1) in vitro and in vivo resulting in higher basal levels of ROS (Reactive Oxygen Species) accumulation. Although decreased TRX1 contributes to higher ROS levels in CR cells, we have found that these CR cells also possess elevate number and active mitochondria as evidence by increased oxygen consumption (4x higher than parental cells) and more intense mitochondria specific fluorescence (mitoTracker) as well as significantly higher mitochondrial membrane potential. These factors could also contribute to higher ROS production. Higher mitochondria activity and ROS also suggest that CR cells may rely more on oxidative metabolism (OXMET). We validated the involvement of specific OXMET components as well as the impact of TRX1 on CR cell metabolism. We examined key proteins in the TCA cycle in SCLC1 (parental), SR2 (resistance counterpart), and TRX1 over-expressing SR2 cells (SR2TRX+). We found that citrate synthetase, the first rate determining enzyme in the TCA cycle, is not changed but ATP-citrate lyase levels exhibit a 3fold increase. Fumarase (FH) which participates in the maintenance of succinate and fumarate equilibrium is increased in SR2 (1.8fold), but attenuates in TRX1 transfected cells, SR2TRX+. This finding implies that SR2 produces more fumarate, a known by-product from urea cycle. We then evaluated argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL), the two key enzymes in urea cycle which generate arginine. While ASL shows no changes, ASS mRNA is 30fold higher in SR2 compares to SCLC1. These results indicate that SCLC1 require an exogenous arginine supply for their growth while SR2 do not. Indeed, we found that SCLC1 could not withstand arginine free media supplement with citrulline (only 30% of cells survive after 48h). In contrast, SR2 which expressed ASS can survive in arginine free media with 80% of cells still viable at 48h. Importantly, over-expression of TRX1 suppresses ASS in SR2 which in turn sensitizes them to arginine deprivation. It is also known that HIF1α negatively regulates ASS and TRX1 is known to increase HIF1α. Thus lower TRX1 found in CR cells most likely increase ASS via decreased HIF1α expression. Here, we found that HIF1α is less in SR2 which may also contribute to decreased glycolysis. To consider this possibility, we assay lactate production and found lower amount of lactic acid in media from CR cells (0.013 vs. 0.009 nM/well/cell, SCLC1 vs. SR2; p=&amp;lt;0.05). Overall, our findings suggest that alteration in tumor metabolism in CR cells is most likely mediated by TRX1 through ROS. These findings may have future clinical application in treating CR patients using agents which generate ROS or target mitochondria. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1138. doi:1538-7445.AM2012-1138</jats:p

Abstract 1428: Targeting metabolic alteration in cisplatin-resistant lung cancer

Abstract We have discovered that all cisplatin resistant (CR) lung cancer cells are less reliant on glycolysis while increasing oxidative metabolism and mitochondrial numbers. Here, we showed that glutamine withdrawal has a detrimental effect on CR cell lines, especially in non small cell lung cancer (NSCLC) CR cells. We assayed for glutaminase, a key enzyme in glutaminolysis pathway and found a significant decrease in protein expression and activity in all our CR cells (n=8, P&amp;lt;0.01). Likewise, NSCLC-CR also take up twice as much L-[G-3H] glutamine when compared to its parental counterpart (n=4, p&amp;lt;0.05). Interestingly, we did not observe significant glutamine uptake in small cell lung cancer (SCLC) CR cells (n=4, P&amp;gt;0.05). Since not all CR cells exhibited similar sensitivity to glutamine withdrawal, we further explored other key amino acids which may be used in TCA cycle. We have found that NSCLC-CR lost argininosuccinate synthetase (ASS) expression while SCLC-CR showed an increase. ASS is a key enzyme in urea cycle to regenerate arginine. Thus, NSCLC-CR will depend on exogenous arginine for their survival. Moreover, arginine in the urea cycle can be used to generate glutamate via proline pathway using P5CDH (pyrroline-5-carboxylate dehydrogenase) which may explain why NSCLC-CR are auxotrophic for both arginine and glutamine. To investigate this possibility, we have studied P5CDH expression in all CR cells and their parental cells in complete and glutamine-free media. We have found that NSCLC-CR has significantly less to non-detectable levels of P5CDH and cannot be upregulated upon glutamine deprivation, while its parental cells have higher P5CDH expression which is further upregulated on glutamine deprivation. In contrast, SCLC-CR has significantly higher P5CDH expression compared to its parental cells and both can be upregulated upon glutamine deprivation. To further confirm this finding, we have overexpressed ASS using the pCMV6 vector containing full length ASS cDNA in NSCLC cells (SCASS). These transfected cells were able to withstand arginine free media compared to the NSCLC-CR transfected with empty vector (10% of NSCLC-CR cells were viable after 72hrs in arginine free media compared to 90% of SCASS were still viable). SCASS also took up less glutamine and was able to withstand glutamine free media when compared to NSCLC-CR. Furthermore, SCASS expressed higher levels of P5CDH and was further upregulated upon glutamine withdrawal. Our data demonstrate a complex metabolic network between arginine, proline, and glutamine. The derangement of the key enzymes which are necessary for generation of these amino acids can be exploited for future clinical application to selectively kill cisplatin resistant cells and improve treatment outcome in lung cancer patients. Supported by Department of Veterans Affairs, CDA2 award. Citation Format: Medhi Wangpaichitr, Chunjing Wu, Ying Ying Li, Shumei Chen, Min You, Vy Dinh, Lynn G. Feun, Macus T. Kuo, Niramol Savaraj. Targeting metabolic alteration in cisplatin-resistant lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1428. doi:10.1158/1538-7445.AM2014-1428</jats:p