Identification and Characterization of a Novel Indoleamine 2,3-Dioxygenase 1 Protein Degrader for Glioblastoma.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a potent immunosuppressive enzyme that inhibits the antitumor immune response through both tryptophan metabolism and non-enzymatic functions. To date, most IDO1-targeted approaches have focused on inhibiting tryptophan metabolism. However, this class of drugs has failed to improve the overall survival of patients with cancer. Here, we developed and characterized proteolysis targeting chimeras (PROTACs) that degrade the IDO1 protein. IDO1-PROTACs were tested for their effects on IDO1 enzyme and non-enzyme activities. After screening a library of IDO1-PROTAC derivatives, a compound was identified that potently degraded the IDO1 protein through cereblon-mediated proteasomal degradation. The IDO1-PROTAC: (i) inhibited IDO1 enzyme activity and IDO1-mediated NF-κB phosphorylation in cultured human glioblastoma (GBM) cells, (ii) degraded the IDO1 protein within intracranial brain tumors in vivo, and (iii) mediated a survival benefit in mice with well-established brain tumors. This study identified and characterized a new IDO1 protein degrader with therapeutic potential for patients with glioblastoma.

Glioblastoma as an age-related neurological disorder in adults.

BACKGROUND: Advanced age is a major risk factor for the development of many diseases including those affecting the central nervous system. Wild-type isocitrate dehydrogenase glioblastoma (IDHwt GBM) is the most common primary malignant brain cancer and accounts for ≥90% of all adult GBM diagnoses. Patients with IDHwt GBM have a median age of diagnosis at 68-70 years of age, and increasing age is associated with an increasingly worse prognosis for patients with this type of GBM. METHODS: The Surveillance, Epidemiology, and End Results, The Cancer Genome Atlas, and the Chinese Glioma Genome Atlas databases were analyzed for mortality indices. Meta-analysis of 80 clinical trials was evaluated for log hazard ratio for aging to tumor survivorship. RESULTS: Despite significant advances in the understanding of intratumoral genetic alterations, molecular characteristics of tumor microenvironments, and relationships between tumor molecular characteristics and the use of targeted therapeutics, life expectancy for older adults with GBM has yet to improve. CONCLUSIONS: Based upon the results of our analysis, we propose that age-dependent factors that are yet to be fully elucidated, contribute to IDHwt GBM patient outcomes.

Tumor Cell IDO Enhances Immune Suppression and Decreases Survival Independent of Tryptophan Metabolism in Glioblastoma.

PURPOSE: Glioblastoma (GBM) is an incurable primary brain tumor that has not benefited from immunotherapy to date. More than 90% of GBM expresses the tryptophan (Trp) metabolic enzyme, indoleamine 2,3-dioxygenase 1 (IDO). This observation supported the historical hypothesis that IDO suppresses the antitumor immune response solely through a mechanism that requires intratumoral Trp depletion. However, recent findings led us to investigate the alternative hypothesis that IDO suppresses the anti-GBM immune response independent of its association with Trp metabolism. EXPERIMENTAL DESIGN: IDO-deficient GBM cell lines reconstituted with IDO wild-type or IDO enzyme-null cDNA were created and validated in vitro and in vivo. Microarray analysis was conducted to search for genes that IDO regulates, followed by the analysis of human GBM cell lines, patient GBM and plasma, and The Cancer Genome Atlas (TCGA) database. Ex vivo cell coculture assays, syngeneic and humanized mouse GBM models, were used to test the alternative hypothesis. RESULTS: Nonenzymic tumor cell IDO activity decreased the survival of experimental animals and increased the expression of complement factor H (CFH) and its isoform, factor H like protein 1 (FHL-1) in human GBM. Tumor cell IDO increased CFH and FHL-1 expression independent of Trp metabolism. Increased intratumoral CFH and FHL-1 levels were associated with poorer survival among patients with glioma. Similar to IDO effects, GBM cell FHL-1 expression increased intratumoral regulatory T cells (Treg) and myeloid-derived suppressor cells while it decreased overall survival in mice with GBM. CONCLUSIONS: Our study reveals a nonmetabolic IDO-mediated enhancement of CFH expression and provides a new therapeutic target for patients with GBM.

Immunosuppressive IDO in Cancer: Mechanisms of Action, Animal Models, and Targeting Strategies.

Indoleamine 2, 3-dioxygenase 1 (IDO; IDO1; INDO) is a rate-limiting enzyme that metabolizes the essential amino acid, tryptophan, into downstream kynurenines. Canonically, the metabolic depletion of tryptophan and/or the accumulation of kynurenine is the mechanism that defines how immunosuppressive IDO inhibits immune cell effector functions and/or facilitates T cell death. Non-canonically, IDO also suppresses immunity through non-enzymic effects. Since IDO targeting compounds predominantly aim to inhibit metabolic activity as evidenced across the numerous clinical trials currently evaluating safety/efficacy in patients with cancer, in addition to the recent disappointment of IDO enzyme inhibitor therapy during the phase III ECHO-301 trial, the issue of IDO non-enzyme effects have come to the forefront of mechanistic and therapeutic consideration(s). Here, we review enzyme-dependent and -independent IDO-mediated immunosuppression as it primarily relates to glioblastoma (GBM); the most common and aggressive primary brain tumor in adults. Our group's recent discovery that IDO levels increase in the brain parenchyma during advanced age and regardless of whether GBM is present, highlights an immunosuppressive synergy between aging-increased IDO activity in cells of the central nervous system that reside outside of the brain tumor but collaborate with GBM cell IDO activity inside of the tumor. Because of their potential value for the in vivo study of IDO, we also review current transgenic animal modeling systems while highlighting three new constructs recently created by our group. This work converges on the central premise that maximal immunotherapeutic efficacy in subjects with advanced cancer requires both IDO enzyme- and non-enzyme-neutralization, which is not adequately addressed by available IDO-targeting pharmacologic approaches at this time.

SOX9 Is Essential for Triple-Negative Breast Cancer Cell Survival and Metastasis.

Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and lacks effective targeted treatment strategies. Previously, we identified 33 transcription factors highly expressed in TNBC. Here, we focused on six sex determining region Y-related HMG-box (SOX) transcription factors (SOX4, 6, 8, 9, 10, and 11) highly expressed in TNBCs. Our siRNA screening assay demonstrated that SOX9 knockdown suppressed TNBC cell growth and invasion in vitro. Thus, we hypothesized that SOX9 is an important regulator of breast cancer survival and metastasis, and demonstrated that knockout of SOX9 reduced breast tumor growth and lung metastasis in vivo. In addition, we found that loss of SOX9 induced profound apoptosis, with only a slight impairment of G1 to S progression within the cell cycle, and that SOX9 directly regulates genes controlling apoptosis. On the basis of published CHIP-seq data, we demonstrated that SOX9 binds to the promoter of apoptosis-regulating genes (tnfrsf1b, fadd, tnfrsf10a, tnfrsf10b, and ripk1), and represses their expression. SOX9 knockdown upregulates these genes, consistent with the induction of apoptosis. Analysis of available CHIP-seq data showed that SOX9 binds to the promoters of several epithelial-mesenchymal transition (EMT)- and metastasis-regulating genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of genes involved in EMT (vim, cldn1, ctnnb1, and zeb1) and that SOX9 knockdown suppresses the expression of these genes. IMPLICATIONS: Our studies identified the SOX9 protein as a "master regulator" of breast cancer cell survival and metastasis, and provide preclinical rationale to develop SOX9 inhibitors for the treatment of women with metastatic triple-negative breast cancer.

Elimination of dormant, autophagic ovarian cancer cells and xenografts through enhanced sensitivity to anaplastic lymphoma kinase inhibition.

BACKGROUND: Poor outcomes for patients with ovarian cancer relate to dormant, drug-resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease. METHODS: By using unbiased small-interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small-molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP-binding protein Di-Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib-induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short-hairpin RNA knockdown of autophagic proteins, and small-molecule inhibitors of STAT3 and BCL-2. RESULTS: Induction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3-expressing tumors treated with crizotinib had significantly decreased tumor burden and long-term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy-mediated apoptosis by decreasing phosphorylated STAT3 and BCL-2 signaling. CONCLUSIONS: Crizotinib may eliminate dormant, autophagic, drug-resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second-look operations should be seriously considered.

A retrospective survival analysis of Glioblastoma patients treated with selective serotonin reuptake inhibitors.

Glioblastoma (GBM) is the most common and aggressive form of malignant glioma in adults with a median overall survival (OS) time of 16-18 months and a median age of diagnosis at 64 years old. Recent work has suggested that depression and psychosocial distress are associated with worse outcomes in patients with GBM. We therefore hypothesized that the targeted neutralization of psychosocial distress with selective serotonin reuptake inhibitor (SSRI) antidepressant treatment would be associated with a longer OS among patients with GBM. To address this hypothesis, we retrospectively studied the association between adjuvant SSRI usage and OS in GBM patients treated by Northwestern Medicine-affiliated providers. The medical records of 497 GBM patients were analyzed after extraction from the Northwestern Medicine Enterprise Data Warehouse. Data were retrospectively studied using a multivariable Cox model with SSRI use defined as a time-dependent variable for estimating the association with OS. Of the 497 patients, 315 individuals died, while 182 were censored due to the loss of follow-up or were alive at the end of our study. Of the 497 patients, 151 had a recorded use of SSRI treatment during the disease course. Unexpectedly, SSRI usage was not associated with an OS effect in both naïve (HR = 0.81, 95% CI = 0.64-1.03) and adjusted time-dependent (HR = 1.26, 95% CI = 0.97-1.63) Cox models. Ultimately, we failed to find an association between SSRI treatment and an improved OS of patients with GBM. Additional work is necessary for understanding the potential therapeutic effects of SSRIs when combined with other treatment approaches, and immunotherapies in particular, for subjects with GBM.

Mutant P53 induces MELK expression by release of wild-type P53-dependent suppression of FOXM1.

Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer, and is associated with a poor prognosis due to frequent distant metastasis and lack of effective targeted therapies. Previously, we identified maternal embryonic leucine zipper kinase (MELK) to be highly expressed in TNBCs as compared with ER-positive breast cancers. Here we determined the molecular mechanism by which MELK is overexpressed in TNBCs. Analysis of publicly available data sets revealed that MELK mRNA is elevated in p53-mutant breast cancers. Consistent with this observation, MELK protein levels are higher in p53-mutant vs. p53 wild-type breast cancer cells. Furthermore, inactivation of wild-type p53, by loss or mutation of the p53 gene, increases MELK expression, whereas overexpression of wild-type p53 in p53-null cells reduces MELK promoter activity and MELK expression. We further analyzed MELK expression in breast cancer data sets and compared that with known wild-type p53 target genes. This analysis revealed that MELK expression strongly correlates with genes known to be suppressed by wild-type p53. Promoter deletion studies identified a p53-responsive region within the MELK promoter that did not map to the p53 consensus response elements, but to a region containing a FOXM1-binding site. Consistent with this result, knockdown of FOXM1 reduced MELK expression in p53-mutant TNBC cells and expression of wild-type p53 reduced FOXM1 expression. ChIP assays demonstrated that expression of wild-type p53 reduces binding of E2F1 (a critical transcription factor controlling FOXM1 expression) to the FOXM1 promoter, thereby, reducing FOXM1 expression. These results show that wild-type p53 suppresses FOXM1 expression, and thus MELK expression, through indirect mechanisms. Overall, these studies demonstrate that wild-type p53 represses MELK expression by inhibiting E2F1A-dependent transcription of FOXM1 and that mutation-driven loss of wild-type p53, which frequently occurs in TNBCs, induces MELK expression by suppressing FOXM1 expression and activity in p53-mutant breast cancers.

The phosphatase PPM1A inhibits triple negative breast cancer growth by blocking cell cycle progression.

Estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and HER2-negative, or "triple negative," breast cancer (TNBC) is a poor prognosis clinical subtype that occurs more frequently in younger women and is commonly treated with toxic chemotherapy. Effective targeted therapy for TNBC is urgently needed. Our previous studies have identified several kinases critical for TNBC growth. Since phosphatases regulate the function of kinase signaling pathways, we sought to identify critical growth-regulatory phosphatases that are expressed differentially in ER-negative, as compared to ER-positive, breast cancers. In this study, we examined the role of one of these differentially expressed phosphatases, the protein phosphatase Mg + 2/Mn + 2 dependent 1A (PPM1A) which is underexpressed in ER-negative breast cancer as compared to ER-positive breast cancers, in regulating TNBC growth. We found that PPM1A is deleted in ~40% of ER-negative breast cancers, and that induced expression of PPM1A suppresses in vitro and in vivo growth of TNBC cells. This study demonstrates that induction of PPM1A expression blocks the cell cycle and reduces CDK and Rb phosphorylation. These results suggest PPM1A is a crucial regulator of cell cycle progression in triple negative breast cancer. Our results also suggest that PPM1A loss should be explored as a predictive biomarker of CDK inhibitor sensitivity.

Targeted reduction of the EGFR protein, but not inhibition of its kinase activity, induces mitophagy and death of cancer cells through activation of mTORC2 and Akt.

The oncogenic epidermal growth factor receptor (EGFR) is commonly overexpressed in solid cancers. The tyrosine kinase activity of EGFR has been a major therapeutic target for cancer; however, the efficacy of EGFR tyrosine kinase inhibitors to treat cancers has been challenged by innate and acquired resistance at the clinic. Accumulating evidence suggests that EGFR possesses kinase-independent pro-survival functions, and that cancer cells are more vulnerable to reduction of EGFR protein than to inhibition of its kinase activity. The molecular mechanism underlying loss-of-EGFR-induced cell death remains largely unknown. In this study, we show that, unlike inhibiting EGFR kinase activity that is known to induce pro-survival non-selective autophagy, downregulating EGFR protein, either by siRNA, or by a synthetic EGFR-downregulating peptide (Herdegradin), kills prostate and ovarian cancer cells via selective mitophagy by activating the mTORC2/Akt axis. Furthermore, Herdegradin induced mitophagy and inhibited the growth of orthotopic ovarian cancers in mice. This study identifies anti-mitophagy as a kinase-independent function of EGFR, reveals a novel function of mTORC2/Akt axis in promoting mitophagy in cancer cells, and offers a novel approach for pharmacological downregulation of EGFR protein as a potential treatment for EGFR-positive cancers.

Molecular Pathways: Targeting Protein Tyrosine Phosphatases in Cancer.

The aberrant activation of oncogenic signaling pathways is a universal phenomenon in cancer and drives tumorigenesis and malignant transformation. This abnormal activation of signaling pathways in cancer is due to the altered expression of protein kinases and phosphatases. In response to extracellular signals, protein kinases activate downstream signaling pathways through a series of protein phosphorylation events, ultimately producing a signal response. Protein tyrosine phosphatases (PTP) are a family of enzymes that hydrolytically remove phosphate groups from proteins. Initially, PTPs were shown to act as tumor suppressor genes by terminating signal responses through the dephosphorylation of oncogenic kinases. More recently, it has become clear that several PTPs overexpressed in human cancers do not suppress tumor growth; instead, they positively regulate signaling pathways and promote tumor development and progression. In this review, we discuss both types of PTPs: those that have tumor suppressor activities as well as those that act as oncogenes. We also discuss the potential of PTP inhibitors for cancer therapy. Clin Cancer Res; 23(9); 2136-42. ©2017 AACR.

Transcriptional regulation of core autophagy and lysosomal genes by the androgen receptor promotes prostate cancer progression.

AR (androgen receptor) signaling is crucial for the development and maintenance of the prostate as well as the initiation and progression of prostate cancer. Despite the AR's central role in prostate cancer progression, it is still unclear which AR-mediated processes drive the disease. Here, we identified 4 core autophagy genes: ATG4B, ATG4D, ULK1, and ULK2, in addition to the transcription factor TFEB, a master regulator of lysosomal biogenesis and function, as transcriptional targets of AR in prostate cancer. These findings were significant in light of our recent observation that androgens promoted prostate cancer cell growth in part through the induction of autophagy. Expression of these 5 genes was essential for maximal androgen-mediated autophagy and cell proliferation. In addition, expression of each of these 5 genes alone or in combination was sufficient to increase prostate cancer cell growth independent of AR activity. Further, bioinformatic analysis demonstrated that the expression of these genes correlated with disease progression in 3 separate clinical cohorts. Collectively, these findings demonstrate a functional role for increased autophagy in prostate cancer progression, provide a mechanism for how autophagy is augmented, and highlight the potential of targeting this process for the treatment of advanced prostate cancer.

Alcohol Regulates Genes that Are Associated with Response to Endocrine Therapy and Attenuates the Actions of Tamoxifen in Breast Cancer Cells.

Hereditary, hormonal, and behavioral factors contribute to the development of breast cancer. Alcohol consumption is a modifiable behavior that is linked to increased breast cancer risks and is associated with the development of hormone-dependent breast cancers as well as disease progression and recurrence following endocrine treatment. In this study we examined the molecular mechanisms of action of alcohol by applying molecular, genetic, and genomic approaches in characterizing its effects on estrogen receptor (ER)-positive breast cancer cells. Treatments with alcohol promoted cell proliferation, increased growth factor signaling, and up-regulated the transcription of the ER target gene GREB1 but not the canonical target TFF1/pS2. Microarray analysis following alcohol treatment identified a large number of alcohol-responsive genes, including those which function in apoptotic and cell proliferation pathways. Furthermore, expression profiles of the responsive gene sets in tumors were strongly associated with clinical outcomes in patients who received endocrine therapy. Correspondingly, alcohol treatment attenuated the anti-proliferative effects of the endocrine therapeutic drug tamoxifen in ER-positive breast cancer cells. To determine the contribution and functions of responsive genes, their differential expression in tumors were assessed between outcome groups. The proto-oncogene BRAF was identified as a novel alcohol- and estrogen-induced gene that showed higher expression in patients with poor outcomes. Knock-down of BRAF, moreover, prevented the proliferation of breast cancer cells. These findings not only highlight the mechanistic basis of the effects of alcohol on breast cancer cells and increased risks for disease incidents and recurrence, but may facilitate the discovery and characterization of novel oncogenic pathways and markers in breast cancer research and therapeutics.

Intracellular activation of EGFR by fatty acid synthase dependent palmitoylation.

Epidermal growth factor receptor (EGFR) is an oncogenic receptor tyrosine kinase. Canonically, the tyrosine kinase activity of EGFR is regulated by its extracellular ligands. However, ligand-independent activation of EGFR exists in certain cancer cells, and the underlying mechanism remains to be defined. In this study, using PC3 and A549 cells as a model, we have found that, in the absence of extracellular ligands, a subpopulation of EGFR is constitutively active, which is needed for maintaining cell proliferation. Furthermore, we have found that fatty acid synthase (FASN)-dependent palmitoylation of EGFR is required for EGFR dimerization and kinase activation. Inhibition of FASN or palmitoyl acyltransferases reduced the activity and down-regulated the levels of EGFR, and sensitized cancer cells to EGFR tyrosine kinase inhibitors. It is concluded that EGFR can be activated intracellularly by FASN-dependent palmitoylation. This mechanism may serve as a new target for improving EGFR-based cancer therapy.

Identification of a Novel Coregulator, SH3YL1, That Interacts With the Androgen Receptor N-Terminus.

Nuclear receptor (NR)-mediated transcriptional activity is a dynamic process that is regulated by the binding of ligands that induce distinct conformational changes in the NR. These structural alterations lead to the differential recruitment of coregulators (coactivators or corepressors) that control the expression of NR-regulated genes. Here, we show that a stretch of proline residues located within the N-terminus of androgen receptor (AR) is a bona fide coregulator binding surface, the disruption of which reduces the androgen-dependent proliferation and migration of prostate cancer (PCa) cells. Using T7 phage display, we identified a novel AR-interacting protein, Src homology 3 (SH3)-domain containing, Ysc84-like 1 (SH3YL1), whose interaction with the receptor is dependent upon this polyproline domain. As with mutations within the AR polyproline domain, knockdown of SH3YL1 attenuated androgen-mediated cell growth and migration. RNA expression analysis revealed that SH3YL1 was required for the induction of a subset of AR-modulated genes. Notable was the observation that ubinuclein 1 (UBN1), a key member of a histone H3.3 chaperone complex, was a transcriptional target of the AR/SH3YL1 complex, correlated with aggressive PCa in patients, and was necessary for the maximal androgen-mediated proliferation and migration of PCa cells. Collectively, these data highlight the importance of an amino-terminal activation domain, its associated coregulator, and downstream transcriptional targets in regulating cellular processes of pathological importance in PCa.

Gain of glucose-independent growth upon metastasis of breast cancer cells to the brain.

Breast cancer brain metastasis is resistant to therapy and a particularly poor prognostic feature in patient survival. Altered metabolism is a common feature of cancer cells, but little is known as to what metabolic changes benefit breast cancer brain metastases. We found that brain metastatic breast cancer cells evolved the ability to survive and proliferate independent of glucose due to enhanced gluconeogenesis and oxidations of glutamine and branched chain amino acids, which together sustain the nonoxidative pentose pathway for purine synthesis. Silencing expression of fructose-1,6-bisphosphatases (FBP) in brain metastatic cells reduced their viability and improved the survival of metastasis-bearing immunocompetent hosts. Clinically, we showed that brain metastases from human breast cancer patients expressed higher levels of FBP and glycogen than the corresponding primary tumors. Together, our findings identify a critical metabolic condition required to sustain brain metastasis and suggest that targeting gluconeogenesis may help eradicate this deadly feature in advanced breast cancer patients.

Involvement of de novo synthesized palmitate and mitochondrial EGFR in EGF induced mitochondrial fusion of cancer cells.

Increased expressions of fatty acid synthase (FASN) and epidermal growth factor receptor (EGFR) are common in cancer cells. De novo synthesis of palmitate by FASN is critical for the survival of cancer cells via mechanisms independent of its role as an energy substrate. Besides the plasma membrane and the nucleus, EGFR can also localize at the mitochondria; however, signals that can activate mitochondrial EGFR (mtEGFR) and the functions of mtEGFR of cancer cells remain unknown. The present study characterizes mtEGFR in the mitochondria of cancer cells (prostate and breast) and reveals that mtEGFR can promote mitochondrial fusion through increasing the protein levels of fusion proteins PHB2 and OPA1. Activation of plasma membranous EGFR (pmEGFR) stimulates the de novo synthesis of palmitate through activation of FASN and ATP-citrate lyase (ACLy). In vitro kinase assay with isolated mitochondria shows that palmitate can activate mtEGFR. Inhibition of FASN blocks the mtEGFR phosphorylation and palmitoylation induced by EGF. Mutational studies show that the cysteine 797 is important for mtEGFR activation and palmitoylation. Inhibition of FASN can block EGF induced mitochondrial fusion and increased the sensitivity of prostate cancer cells to EGFR tyrosine kinase inhibitor. In conclusion, these results suggest that mtEGFR can be activated by pmEGFR through de novo synthesized palmitate to promote mitochondrial fusion and survival of cancer cells. This mechanism may serve as a novel target to improve EGFR-based cancer therapy.

Antiproliferative effects and mechanisms of liver X receptor ligands in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) is difficult to detect early and is often resistant to standard chemotherapeutic options, contributing to extremely poor disease outcomes. Members of the nuclear receptor superfamily carry out essential biological functions such as hormone signaling and are successfully targeted in the treatment of endocrine-related malignancies. Liver X receptors (LXRs) are nuclear receptors that regulate cholesterol homeostasis, lipid metabolism, and inflammation, and LXR agonists have been developed to regulate LXR function in these processes. Intriguingly, these compounds also exhibit antiproliferative activity in diverse types of cancer cells. In this study, LXR agonist treatments disrupted proliferation, cell-cycle progression, and colony-formation of PDAC cells. At the molecular level, treatments downregulated expression of proteins involved in cell cycle progression and growth factor signaling. Microarray experiments further revealed changes in expression profiles of multiple gene networks involved in biological processes and pathways essential for cell growth and proliferation following LXR activation. These results establish the antiproliferative effects of LXR agonists and potential mechanisms of action in PDAC cells and provide evidence for their potential application in the prevention and treatment of PDAC.

ATP citrate lyase mediates resistance of colorectal cancer cells to SN38.

Combination chemotherapy is standard for metastatic colorectal cancer; however, nearly all patients develop drug resistance. Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may enable identification of novel targets and more effective therapy. Irinotecan is commonly used in first- and second-line therapy for patients with metastatic colorectal cancer, with the active metabolite being SN38. Emerging evidence suggests that altered metabolism in cancer cells is fundamentally involved in the development of drug resistance. Using Oncomine and unbiased proteomic profiling, we found that ATP citrate lyase (ACLy), the first-step rate-limiting enzyme for de novo lipogenesis, was upregulated in colorectal cancer compared with its levels in normal mucosa and in chemoresistant colorectal cancer cells compared with isogenic chemo-naïve colorectal cancer cells. Overexpression of exogenous ACLy by lentivirus transduction in chemo-naïve colorectal cancer cells led to significant chemoresistance to SN38 but not to 5-fluorouracil or oxaliplatin. Knockdown of ACLy by siRNA or inhibition of its activity by a small-molecule inhibitor sensitized chemo-naïve colorectal cancer cells to SN38. Furthermore, ACLy was significantly increased in cancer cells that had acquired resistance to SN38. In contrast to chemo-naïve cells, targeting ACLy alone was not effective in resensitizing resistant cells to SN38, due to a compensatory activation of the AKT pathway triggered by ACLy suppression. Combined inhibition of AKT signaling and ACLy successfully resensitized SN38-resistant cells to SN38. We conclude that targeting ACLy may improve the therapeutic effects of irinotecan and that simultaneous targeting of ACLy and AKT may be warranted to overcome SN38 resistance.

EGFR-SGLT1 interaction does not respond to EGFR modulators, but inhibition of SGLT1 sensitizes prostate cancer cells to EGFR tyrosine kinase inhibitors.

BACKGROUND: Overexpression of epidermal growth factor receptor (EGFR) is associated with poor prognosis in malignant tumors. Sodium/glucose co-transporter 1 (SGLT1) is an active glucose transporter that is overexpressed in many cancers including prostate cancer. Previously, we found that EGFR interacts with and stabilizes SGLT1 in cancer cells. METHODS: In this study, we determined the micro-domain of EGFR that is required for its interaction with SGLT1 and the effects of activation/inactivation of EGFR on EGFR-SGLT1 interaction, measured the expression of EGFR and SGLT1 in prostate cancer tissues, and tested the effect of inhibition of SGLT1 on the sensitivity of prostate cancer cells to EGFR tyrosine inhibitors. RESULTS: We found that the autophosphorylation region (978-1210 amino acids) of EGFR was required for its sufficient interaction with SGLT1 and that this interaction was independent of EGFR's tyrosine kinase activity. Most importantly, the EGFR-SGLT1 interaction does not respond to EGFR tyrosine kinase modulators (EGF and tyrosine kinase inhibitors). EGFR and SGLT1 co-localized in prostate cancer tissues, and inhibition of SGLT1 by a SGLT1 inhibitor (Phlorizin) sensitized prostate cancer cells to EGFR inhibitors (Gefitinib and Erlotinib). CONCLUSION: These data suggest that EGFR in cancer cells can exist as either a tyrosine kinase modulator responsive status or an irresponsive status. SGLT1 is a protein involved in EGFR's functions that are irresponsive to EGFR tyrosine kinase inhibitors and, therefore, the EGFR-SGLT1 interaction might be a novel target for prostate cancer therapy.