Rationale : Cancer cells reprogram cellular metabolism to fulfill their needs for rapid growth and metastasis. However, the mechanism controlling this reprogramming is poorly understood. We searched for upregulated signaling in metastatic colorectal cancer and investigated the mechanism by which Glut3 promotes tumor metastasis. Methods : We compared RNA levels and glycolytic capacity in primary and metastatic colon cancer. The expression and association of Glut3 with clinical prognosis in colon cancer tissues was determined by immunohistochemistry. Glut3 gain-of-function and loss-of-function were established using colon cancer HCT116, HT29, and metastatic 116-LM cells, and tumor invasiveness and stemness properties were evaluated. Metabolomic profiles were analyzed by GC/MS and CE-TOF/MS. The metastatic burden in mice fed a high-fat sucrose diet was assessed by intravenous inoculation with Glut3 knockdown 116-LM cells. Results : Upregulation of glycolytic genes and glycolytic capacity was detected in metastatic colorectal cancer cells. Specifically, Glut3 overexpression was associated with metastasis and poor survival in colorectal cancer patients. Mechanistically, Glut3 promoted invasiveness and stemness in a Yes-associated protein (YAP)-dependent manner. Activation of YAP in turn transactivated Glut3 and regulated a group of glycolytic genes. Interestingly, the expression and phosphorylation of PKM2 were concomitantly upregulated in metastatic colorectal cancer, and it was found to interact with YAP and enhance the expression of Glut3. Importantly, a high-fat high-sucrose diet promoted tumor metastasis, whereas the inhibition of either Glut3 or YAP effectively reduced the metastatic burden. Conclusion : Activation of the Glut3-YAP signaling pathway acts as a master activator to reprogram cancer metabolism and thereby promotes metastasis. Our findings reveal the importance of metabolic reprogramming in supporting cancer metastasis as well as possible therapeutic targets.

Arginine synthesis deficiency due to the suppressed expression of ASS1 (argininosuccinate synthetase 1) represents one of the most frequently occurring metabolic defects of tumor cells. Arginine-deprivation therapy has gained increasing attention in recent years. One challenge of ADI-PEG20 (pegylated ADI) therapy is the development of drug resistance caused by restoration of ASS1 expression and other factors. The goal of this work is to identify novel factors conferring therapy resistance. Methods: Multiple, independently derived ADI-resistant clones including derivatives of breast (MDA-MB-231 and BT-549) and prostate (PC3, CWR22Rv1, and DU145) cancer cells were developed. RNA-seq and RT-PCR were used to identify genes upregulated in the resistant clones. Unbiased genome-wide CRISPR/Cas9 knockout screening was used to identify genes whose absence confers sensitivity to these cells. shRNA and CRISPR/Cas9 knockout as well as overexpression approaches were used to validate the functions of the resistant genes both in vitro and in xenograft models. The signal pathways were verified by western blotting and cytokine release. Results: Based on unbiased CRISPR/Cas9 knockout screening and RNA-seq analyses of independently derived ADI-resistant (ADIR) clones, aberrant activation of the TREM1/CCL2 axis in addition to ASS1 expression was consistently identified as the resistant factors. Unlike ADIR, MDA-MB-231 overexpressing ASS1 cells achieved only moderate ADI resistance both in vitro and in vivo , and overexpression of ASS1 alone does not activate the TREM1/CCL2 axis. These data suggested that upregulation of TREM1 is an independent factor in the development of strong resistance, which is accompanied by activation of the AKT/mTOR/STAT3/CCL2 pathway and contributes to cell survival and overcoming the tumor suppressive effects of ASS1 overexpression. Importantly, knockdown of TREM1 or CCL2 significantly sensitized ADIR toward ADI. Similar results were obtained in BT-549 breast cancer cell line as well as castration-resistant prostate cancer cells. The present study sheds light on the detailed mechanisms of resistance to arginine-deprivation therapy and uncovers novel targets to overcome resistance. Conclusion: We uncovered TREM1/CCL2 activation, in addition to restored ASS1 expression, as a key pathway involved in full ADI-resistance in breast and prostate cancer models.

Reactive oxygen species (ROS) homeostasis is maintained at a higher level in cancer cells, which promotes tumorigenesis. Oxidative stress induced by anticancer drugs may further increase ROS to promote apoptosis, but can also enhance the metastasis of cancer cells. The effects of ROS homeostasis on cancer cells remain to be fully elucidated. In the present study, the effect of a reduction in manganese superoxide dismutase (MnSOD) on the migration and invasion of A431 cells was investigated. Our previous micro-assay data revealed that the mRNA expression of MnSOD was higher in the invasive A431-III cell line compared with that in the parental A431 cell line (A431-P). In the present study, high protein levels of MnSOD and H 2 O 2 production were observed in A431-III cells; however, catalase protein levels were significantly lower in A431-III cells compared with those in the A431-P cell line. The knockdown of MnSOD increased H 2 O 2 levels, enzyme activity, the mRNA levels of matrix metalloproteinase-1, -2 and -9, and the migratory and invasive abilities of the cells. Inducing a reduction in H 2 O 2 using diphenyleneiodonium (DPI) and N-acetyl-l-cysteine decreased the migratory abilities of the cell lines, and DPI attenuated the migratory ability that had been increased by MnSOD small interfering RNA knockdown. Luteolin (Lu) and quercetin (Qu) increased the expression of catalase and reduced H 2 O 2 levels, but without an observed change in the protein levels of MnSOD. Taken together, these data suggest that reduced MnSOD may induce ROS imbalance in cells and promote the metastatic ability of cancer cells. Lu and Qu may attenuate these processes and may be promising potential anticancer agents.

This study demonstrated for the first time that curcumin effectively inhibits the growth of triple-negative breast cancer (TNBC) tumors by inhibiting the expression of salt-induced kinase-3 (SIK3) protein in patient-derived xenografted tumor mice (TNBC-PDX). For TNBC patients, chemotherapy is the only option for postoperative adjuvant treatment. In this study, we detected the SIK3 mRNA expression in paired-breast cancer tissues by qPCR analysis. The results revealed that SIK3 mRNA expression was significantly higher in tumor tissues when compared to the normal adjacent tissues (73.25 times, n = 183). Thus, it is proposed for the first time that the antitumor effect induced by curcumin by targeting SIK3 can be used as a novel strategy for the therapy of TNBC tumors. In vitro mechanism studies have shown that curcumin (>25 μM) inhibits the SIK3-mediated cyclin D upregulation, thereby inhibiting the G1/S cell cycle and arresting TNBC (MDA-MB-231) cancer cell growth. The SIK3 overexpression was associated with increased mesenchymal markers (i.e., Vimentin, α-SMA, MMP3, and Twist) during epithelial–mesenchymal transition (EMT). Our results demonstrated that curcumin inhibits the SIK3-mediated EMT, effectively attenuating the tumor migration. For clinical indications, dietary nutrients (such as curcumin) as an adjuvant to chemotherapy should be helpful to TNBC patients because the current trend is to shrink the tumor with preoperative chemotherapy and then perform surgery. In addition, from the perspective of chemoprevention, curcumin has excellent clinical application value.

There is overwhelming evidence that tyrosine kinases play an important role in cancer development. As a prototype of targeted therapy, tyrosine kinase inhibitors are now successfully applied to cancer treatment. However, as single agents, tyrosine kinase inhibitors have not achieved satisfactory results in the treatment of prostate cancer, principally due to their inability to efficiently kill tumor cells. Our lab has been interested in the role of the Src complex in prostate cancer progression including the induction of androgen independence and metastasis. Previously, we reported that Src inhibitors such as saracatinib and PP2 caused G1 growth arrest and diminished invasiveness in prostate cancer cells, but rarely apoptosis. Here, we have shown that Src family kinase (SFK) inhibitors can induce a high level of autophagy, which protects treated cells from undergoing apoptosis. Src siRNA knockdown experiments confirmed that autophagy was indeed caused by the lack of Src activity. The SFK inhibitor-induced autophagy is accompanied by the inhibition of PI3K (type I)/Akt/mTOR signaling pathway. To test whether autophagy blockade could lead to enhanced cell death, pharmacological inhibitors (3-methyladenine and chloroquine) and a genetic inhibitor (siRNA targeting Atg7) were used in combination with SFK inhibitors. The results showed that autophagy inhibition effectively enhanced cell killing induced by SFK inhibitors. Importantly, we showed a combination of saracatinib with chloroquine in mice significantly reduced prostate cancer (PC3) xenograft growth, compared with the control group. Taken together, these data suggest that 1) autophagy serves a protective role in SFK inhibitor-mediated cell killing; 2) clinically acceptable autophagy modulators may be used beneficially as adjunctive therapeutic agents for SFK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4684.