Background and Objectives: Cancer, as the second leading cause of death in the United States, poses a huge healthcare burden. Barriers to access to advanced therapies influence the outcome of cancer treatment. In this study, we examined whether insurance types affect the quality of cancer clinical care. Materials and Methods: Data for 13,340 cancer patients with Purchased or Medicaid insurance from the All of Us database were collected for this study. The chi-squared test of proportions was employed to determine the significance of patient cohort characteristics and the accessibility of healthcare services between the Purchased and Medicaid insurance groups. Results: Cancer patients who are African American, with lower socioeconomic status, or with lower educational attainment are more likely to be insured by Medicaid. An analysis of the survey questions demonstrated the relationship between income and education level and insurance type, as Medicaid cancer patients were less likely to receive primary care and specialist physician access and more likely to request lower-cost medications. Conclusions: The inequities of the US healthcare system are observed for cancer patient care; access to physicians and medications is highly varied and dependent on insurance types. Socioeconomic factors further influence insurance types, generating a significant impact on the overall clinical care quality for cancer patients that eventually determines treatment outcomes and the quality of life.

Background. Deficiency of interleukin-1 receptor antagonist (DIRA) is a rare life-threatening autosomal recessive autoinflammatory disease with symptoms including but not limited to osteomyelitis, periostitis, and systemic inflammation. DIRA is developed from the loss-of-function biallelic mutations of the IL1RN gene that encodes IL-1 receptor antagonist (IL-1RA), leading to the unchecked pro-inflammatory signaling and subsequent systemic inflammation. Thus, anakinra as the recombinant IL-1RA has become the primary drug to treat DIRA. Although anakinra has been effective for the complete remission of DIRA, it has also shown various side effects. To confirm the efficacy and safety issues associated with DIRA treatment, we conducted a literature review and secondary data analysis to enhance our understanding on this important topic. Methods. Through comprehensive literature search, we have identified 15 papers with 25 patients studied. The demographic, clinical, and genetic data were extracted, followed by statistical analysis to support the physiological mechanisms of anakinra treatment. Results. Through the literature review and data analysis, it was found that 88% of patients had complete clinical remission of DIRA upon continual treatment with anakinra; patients had a mean improvement of Hemoglobin (+3.18 g/dL), Erythrocyte Sedimentation Rate (−53.4 mm/h), and C-reactive Protein (−135.45 mg/L) levels, suggesting that the improvement of hematopoietic function and inflammation is a mechanism for anakinra treatment. Various genetic variants were also identified from the patient data that cause the loss of function of IL-1RA, providing real patient genomic data to support the anakinra treatment. Conclusions. Considering the inconsistency and certain variations from clinical research influenced by specific conditions, this review along with the data analysis confirms the efficacy and safety of anakinra treatment for DIRA.

Background and Objectives: Cancer as the second leading cause of death in the United States poses a huge healthcare burden. Barriers to access to advanced therapies influence the outcome of cancer treatment. In this study, we examined whether insurance types affect the quality of cancer clinical care regarding access to an important monoclonal antibody, ramucirumab. Materials and Methods: Data for 13,340 cancer patients with Purchased or Medicaid insurance from the All of Us Database were collected for this study. The Chi-square test of proportions was employed to determine the significance of patient cohort characteristics and ramucirumab usage between Purchased and Medicaid insurance groups. The independent t-test was utilized to assess the influences of other determinants on insurance types. Results: Cancer patients who are African American, with lower socioeconomic status, or with lower educational attainment are more likely to be insured by Medicaid. Analysis of survey questions demonstrated the relationship between income and education level with insurance type as Medicaid cancer patients were less likely to receive primary care and specialist physician access and more likely to request lower cost medications. In addition, those with Medicaid insurance were identified to have poorer access to expensive therapeutics like ramucirumab compared to those with Purchased insurance. Conclusions: The inequities of the US healthcare system are observed for cancer patient care; access to physicians and therapeutics are highly varied and dependent on insurance types. Socioeconomic status determines insurance type, which unfortunately generates a significant impact on cancer treatment and disease outcome.

Triple negative breast cancer (TNBC) is frequently diagnosed in younger women and is prevalent in African American women. Most TNBCs are highly aggressive with early pattern of metastasis, and with limited treatment options and poor prognosis. Finding effective therapeutic targets for TNBC has proven difficult so far. Therefore, there is an urgent need to develop new and novel therapeutic management of these patients. Recent studies have shown that glucocorticoid receptor (GR) is a mediator of pro-survival genes in TNBC cells, and its expression is predictive of increased risk of metastasis in TNBC tumors and decreased rate of patient survival. Therefore, blocking the effects of GR should be beneficial for TNBC patients. However, GR antagonists such as RU486 (mifepristone) targeting AF2/coactivators have shown only partial success in TNBC cell lines as well as in animal models. Based on our previous data we hypothesize that since in TNBC cells, the GR is constitutively active; therefore, attempts to block/inhibit GR activity through classic antagonists such as mifepristone that bind to ligand binding pocket of the receptor will have a limited clinical efficacy and higher side effects. To circumvent this problem, in this study, we propose a novel and innovative therapeutic approach to target constitutively active AF1 domain of the GR, which exists in an intrinsically disordered (ID) conformation. In this study, we tested our hypothesis that inhibiting/blocking TATA-box binding protein (TBP)-mediated GR’s AF1 activity by our newly identified molecule, PepT will attenuate TNBC cell survival via altering associated endogenous gene expressions. Our binding kinetics of AF1 showed that PepT binds to AF1 with about 10-fold higher affinity when compared with TBP’s binding to AF1. AF1 pre-bound with PepT fails to bind to TBP suggesting that PepT competes and blocks AF1-TBP interaction. We also found that treatment of MDA-MB-231 cells with PepT results in significant attenuation of cell viability and proliferation. Together, these results support our hypothesis that PepT has potential to kill TNBC cells by blocking GR AF1:co-regulator interactions. Citation Format: Shagufta H. Khan, Christian Carbe, Jun Ling, Raj Kumar. A novel mechanism for therapeutic targeting of the glucocorticoid receptor in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3063.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection has claimed millions of lives since late 2019, yet there are still many unexplored areas in its pathogenesis and clinical outcomes. COVID-19 is a disease that can affects multiple systems, some of which are overlapped with those modulated by gut microbiota, especially the immune system, thus leading to our concentration on analyzing the roles of microbiota in COVID-19 pathogenesis through the gut-lung axis. Dysbiosis of the commensal intestinal microbes and their metabolites (e.g., SCFAs) as well as the expression and activity of ACE2 in the gut could influence the host's immune system in COVID-19 patients. Moreover, it has been known that the elderly and individuals diagnosed with comorbidities (e.g., hypertension, type 2 diabetes mellitus, cardiovascular disease, etc.) are more susceptible to gut flora alterations, SARS-CoV-2 infection, and death. Thus, in this review we will focus on analyzing how the gut microbiota regulates the immune system that leads to different responses to SARS-CoV-2 infection. Since diet is a major factor to modulate the status of gut microbiota, dietary influence on COVID-19 pathogenesis will be also discussed, aiming to shed light on how diet-modulated gut microbiota regulates the susceptibility, severity, and treatment of SARS-CoV-2 infection.

Abstract eIF4A3 is a unique eukaryotic translation initiation factor (eIF) with broad functions in the whole process of RNA metabolism, including mRNA splicing via exon junction complex (EJC), mRNA nuclear export, translation, and RNA surveillance through nonsense-mediated RNA decay (NMD). These post-transcriptional processes are highly involved in cancer development. However, the roles of eIF4A3 in cancers have not addressed. In this study, by analyzing a breast cancer patient database, we identified that the up-regulation of eIF4A3 is correlated with poor survival of triple negative breast cancer (TNBC) patients. Overexpression of eIF4A3, along with other EJC components (Magoh, Y14 and CASC3), were observed in multiple TNBC cell lines as compared to human normal cells and mouse normal tissues. Knockdown of eIF4A3 by RNAi was found to strongly inhibit TNBC cell proliferation in vitro; down-regulation of endogenous eIF4A3 also suppressed tumor growth in a mouse model. Dominant negative eIF4A3 and an ATP-binding defective mutants inhibited TNBC cell growth in the clonogenic assay. Furthermore, TNBC cells were found to be more sensitive to eIF4A3-specific inhibitors than luminal A and B subtypes of breast cancer cells. Thus, our study elucidates a strong cancer-promoting activity of eIF4A3 in breast cancer growth at cellular and animal model levels, warranting a new strategy to target eIF4A3 for breast cancer therapy, especially for TNBC as the most difficult subtype to treat. Citation Format: Jun Ling, Allen Huang, Ai Ohno, Joe Chen, Maria Cavataio. Targeting eIF4A3 as a multifunctional translation initiation factor for breast cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2461.

p21-activated protein kinase (PAK2) is a unique member of the PAK family kinases that plays important roles in stress signaling. It can be activated by binding to the small GTPase, Cdc42 and Rac1, or by caspase 3 cleavage. Cdc42-activated PAK2 mediates cytostasis, whereas caspase 3-cleaved PAK2 contributes to apoptosis. However, the relationship between these two states of PAK2 activation remains elusive. In this study, through protein biochemical analyses and various cell-based assays, we demonstrated that full-length PAK2 activated by Cdc42 was resistant to the cleavage by caspase 3 in vitro and within cells. When mammalian cells were treated by oxidative stress using hydrogen peroxide, PAK2 was highly activated through caspase 3 cleavage that led to apoptosis. However, when PAK2 was pre-activated by Cdc42 or by mild stress such as serum deprivation, it was no longer able to be cleaved by caspase 3 upon hydrogen peroxide treatment, and the subsequent apoptosis was also largely inhibited. Furthermore, cells expressing active mutants of full-length PAK2 became more resistant to hydrogen peroxide-induced apoptosis than inactive mutants. Taken together, this study identified two states of PAK2 activation, wherein Cdc42- and autophosphorylation-dependent activation inhibited the constitutive activation of PAK2 by caspase cleavage. The regulation between these two states of PAK2 activation provides a new molecular mechanism to support PAK2 as a molecular switch for controlling cytostasis and apoptosis in response to different types and levels of stress with broad physiological and pathological relevance.

HSP90 (heat shock protein 90) is a central chaperone to regulate protein folding for a large array of client proteins as important drivers for cancer development. Its overexpression in bladder cancer, especially the most aggressive muscle-invasive subtype (MIBC), attracts a lot of attention for drug development. Although many HSP90 inhibitors have been tested in clinical trials for many cancers, none of them has been approved by FDA for cancer therapy. One of critical problems is the co-activation of heat shock response (HSR), an integrated stress response pathway with strong pro-survival function. Our previous studies also observed the robust activation of HSF1 (an effector of HSR) and HSP70 (a target gene of HSF1) associated with the inhibition of HSP90 by its N-terminal (NT) inhibitors, thereby impairing the effect of HSP90 NT inhibitor. Intriguingly, HSP90 inhibitors tested in the clinical trials so far are all NT inhibitors, further promoting us to explore the HSP90 C-terminal (CT) inhibitors as a new strategy for MIBC therapy. In this study, a number of MIBC cell lines representing different cancer stages were utilized to generate more physiologically relevant data. When UMUC3, T24, SW-780, and J82 cells were treated by novobiocin (Novo, a HSP90 CT inhibitor) at a series of concentrations, it was found that Novo inhibited the proliferation of all cells. However, their sensitivities to Novo were different in the following order: T24> SW-780> UMUC3> J82. This effect was partially correlated with the aggressiveness of cancer stage, wherein higher grade MIBC cells are less sensitive to Novo treatment. Western blotting (WB) analysis indicated that HSF1 and HSP70 were not activated. More importantly, client kinase AKT1 was highly activated in SW-780 but not in all other three cells; ERK1/2 was highly activated in UMUC3 but not in all other three cells, suggesting that the activation of kinases is differential and cell type-dependent. Confocal fluorescence microscopic analysis revealed that HSP90 cellular localization was also altered by Novo from more diffused cytoplasmic distribution to more perinuclear localization, whereas the predominant nuclear localization of HSF1 was not changed by Novo treatment, suggesting the localization of HSP90, different modified forms, or the interaction with other proteins may also contribute to HSP90’s functions in cell growth. These aspects are under investigation by various biochemical and cell biology techniques to validate the advantage of HSP90 CT inhibitors for MIBC therapy. (* corresponding authors) Note: This abstract was not presented at the meeting. Citation Format: Jun Ling, Connor Magura, Vaibhav Sharma, Maisara Rahimi, Heinric Williams. Selective inhibition of HSP90 by its C-terminal inhibitors is more effective to suppress bladder cancer cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2660.

Background Dietary salts sodium (Na+), potassium (K+), magnesium (Mg2+), and calcium (Ca2+) are important in metabolic diseases. Yet, we do not have sufficient understanding on the salts global molecular network in these diseases. In this systematic review we have pooled information to identify the general effect of salts on obesity, insulin resistance and hypertension. Aims To assess the roles of salts in metabolic disorders by focusing on their individual effect and the network effect among these salts. Methods We searched articles in PubMed, EMBASE and Google Scholar. We selected original laboratory research, systematic reviews, clinical trials, observational studies and epidemiological data that focused on dietary salts and followed the preferred reporting items for systematic review in designing the present systematic review. Results From the initial search of 2898 studies we selected a total of 199 articles that met our inclusion criteria and data extraction. Alterations in metabolic pathways associated with the sensitivity of sodium, potassium, magnesium and calcium may lead to obesity, hypertension, and insulin resistance. We found that the results of most laboratory research, animal studies and clinical trials are coherent but some research outcome are either inconsistent or inconclusive. Conclusion Important of salts in metabolic disorder is evident. In order to assess the effects of dietary salts in metablic diseases, environmental factors, dietary habits, physical activity, and the microbiome, should be considered in any study. Although interest in this area of research continues to grow, the challenge is to integrate the action of these salts in metabolic syndrom.

Abstract Steroid hormone receptors such as estrogen and progesterone receptors are well studied in breast cancer pathology; they are also used as drug targets for breast cancer therapy in the clinic. In contrast, glucocorticoid (GC) as a ubiquitous stress activated steroid hormone is less investigated in breast cancer. However, GC is frequently used as a co-treatment for breast cancer chemotherapy that generates some controversial effects to even promote cancer progression or recurrence in certain subtypes of breast cancer. To address this clinical issue, we focused on investigating the specificity of GC signaling on breast cancer cell behaviors in this study. When breast cancer cells were treated by dexamethasone (Dex), they were more responsive to cell migration (measured by electric cell-substrate impedance sensing, ECIS) than the effects on cell proliferation and apoptosis, wherein MDA-231 (triple negative breast cancer) cell was more sensitive to Dex than MCF7 cell (luminal A subtype). Further gene expression analysis by qRT-PCR microarray revealed that the glucocorticoid receptor (GR) responsive gene patterns are different between these two subtypes of breast cancer cells. One of remarked changes was that Snai2 (a zinc finger transcriptional factor) was highly activated in both cell lines. Western blotting analysis confirmed that MDA-231 cells have much higher basal level of Snai2 but with less fold increase upon Dex treatment than MCF-7. GR response elements (GREs) were also identified in the promoter region of Snai2, thus confirming Snai2 as a new target gene of GR in breast cancer. When Snai2 was knocked out by CRISPR, it was found that the basal migration rate was decreased in both cell lines. More importantly, their response rates to Dex treatment were also decreased as compared to the wild type cells. These decreases in cell migration are similar to those treated by GR antagonist RU486, thus confirming that Snai2 is a mediator to regulate the effects of GC/GR signaling on breast cancer cell migration. In summary, this study identified Snai2 as a new target gene of GR to regulate cell migration and potentially metastasis in response to GC signaling. The differential regulation of different subtypes of breast cancer cells by GC signaling was also confirmed. (+ equal contribution; * Corresponding author). Citation Format: Jun Ling, Adit Singhal, Zenaida P. Lopez-Dee, Brittany Porreca, Trinity Sprague. Snai2 is a new target to mediate glucocorticoid signaling on breast cancer cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 45.

Although the incidence rate of pancreatic cancer is higher in men than in women, Oral Contraceptives (OC) and Hormone Replacement Therapy (HRT) commonly used in women may constitute a risk or protective factor for pancreatic cancer. Some studies have examined these relationships, but no coherent conclusions have been reached. Thus, we conducted a meta-analysis in this study by extensive search of literature using PubMed and EMBASE databases. It was found that OC use was associated with the significant decrease in pancreatic cancer risk (pooled OR 0.75, 95% CI 0.61-0.91). Subgroup analysis identified that OC decreased the risk in the pancreatic cancer population-based studies (pooled OR 0.76, 95 % CI 0.67-0.86) but was controversial in the hospital-based studies (pooled OR 1.21, 95% CI 0.94-1.57). HRT did not show the significant effect on pancreatic cancer risk (pooled OR 1.00, 95 % CI 0.79-1.26). However, subgroup analysis identified the decreased risk in North America population (pooled OR 0.81, 95% CI 0.65-1.01) but the increased risk in Europe population (pooled OR 1.51, 95% CI 1.16-1.97). In conclusion, OC use was demonstrated to have a protective effect against pancreatic cancer in women, where as HRT showed no effect or diverse effects in different populations. Our results increase the awareness of using HRT in women when considering pancreatic cancer risk. The subgroup analysis also suggests that more clinical studies are needed to clarify the specific relationship between OC or HRT and pancreatic cancer risk in different populations.

Dietary salts are important factors in metabolic disorders. They are vital components of enzymes, vitamins, hormones, and signal transduction that act synergistically to regulate lipid metabolism. Our previous studies have identified that Krüppel-like factor −3 (KLF-3) is an essential regulator of lipid metabolism. However, it is not known if KLF-2 also regulates lipid metabolism and whether KLF-2 and −3 mediate the effects of dietary salts on lipid metabolism.

Background: Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis-a chronic, painful bladder disease. APF inhibits the proliferation of normal bladder epithelial cells and cancer cells in vitro, presumably by binding to its cellular receptor, cytoskeleton associated-protein 4 (CKAP4); however, the biophysical interaction of APF with CKAP4 has not been characterized previously. In this study, we used surface plasmon resonance (SPR) to explore the binding kinetics of the interaction of APF and as-APF (a desialylated APF analogue with full activity) to CKAP4.Results: We immobilized non-glycosylated APF (TVPAAVVVA) to the Fc1 channel as the control and as-APF to Fc2 channel as the ligand in order to measure the binding of CKAP4 recombinant proteins encompassing only the extracellular domain (Aa 127-602) or the extracellular domain plus the transmembrane domain (Aa 106-602). Positive binding was detected to both CKAP4(126-602) and CKAP4(106-602), suggesting that as-APF can bind specifically to CKAP4 and that the potential binding site(s) are located within the extracellular domain. To identify the primary APF binding site(s) within the CKAP4 extracellular domain, deletion mutants were designed according to structural predictions, and the purified recombinant proteins were immobilized on a CM5 chip through amine-coupling to measure as-APF binding activity. Importantly, both CKAP4(127-360) and CKAP4(361-524) exhibited a fast association rate (k(on)) and a slow dissociation rate (k(off)), thus generating high binding affinity and suggesting that both regions contribute relatively equally to overall as-APF binding. Therefore, two or more as-APF binding sites may exist within the Aa 127-524 region of the CKAP4 extracellular domain.Conclusions: We determined that the CKAP4(127-360) and CKAP4(361-524) mutants exhibit improved binding activity to as-APF as compared to the full-length extracellular domain, making it possible to detect low concentrations of as-APF in urine, thereby establishing a foundation for a non-invasive diagnostic assay for IC. Further, these data have revealed novel APF binding site(s) suggesting that targeting this region of CKAP4 to inhibit APF binding may be a useful strategy for treating IC-related bladder pathology.

Phthalates are a group of plasticizers that are widely used in many consumer products and medical devices, thus generating a huge burden to human health. Phthalates have been known to cause a number of developmental and reproductive disorders functioning as endocrine modulators. They are also involved in carcinogenesis with mechanisms less understood. To further understand the molecular mechanisms of phthalate toxicity, in this study we reported a new effect of phthalates on mRNA translation/protein synthesis, a key regulatory step of gene expression. Butyl benzyl phthalate (BBP) was found to directly inhibit mRNA translation in vitro but showed a complicated pattern of affecting mRNA translation in cells. In human kidney embryonic cell (HEK-293T), BBP increased cap-dependent mRNA translation at lower concentrations but showed inhibitory effect at higher concentrations. Cap-independent translation was not affected. On the other hand, mono (2-ethylhexyl) phthalate (MEHP) as a major metabolite of another important phthalate di (2-ethylhexyl) phthalate (DEHP) inhibited both can-dependent and -independent mRNA translation in vivo. In contrast, BBP and MEHP exhibited an overall promoting effect on mRNA translation in cancer cells. Mechanistic studies identified that the level and phosphorylation of eIF4E-BP (eIF4E binding protein) and the amount of eIF4GI in eIF4F complex were altered in accordance with the effect of BBP on translation. BBP was also identified to directly bind to eIF4E, providing a further mechanism underlying the regulation of mRNA by phthalate. At the cellular level BBP inhibited normal cell growth but slightly promoted cancer cells (HT29) growth. Overall, this study provides the first evidence that phthalates can directly regulate mRNA translation as a novel mechanism to mediate their biological toxicities.

e19058 Background: The use of radiation therapy (RT) in the treatment of DLBCL is a topic of debate that continues to evolve as clinical trials reveal the heterogeneous nature of the disease. Molecular profiling has classified DLBCL into germinal center B cell-like (GCB) and activated B cell-like (ABC) subtypes that show remarkable differences in treatment response and outcomes with standard chemotherapy. Response to RT in DLBCL subtypes has yet to be determined; however, may provide insight into who should receive treatment. Methods: Four DLBCL cell lines: WSU-DLCL2 and SUDHL-6 (GCB); Riva and SUDHL-2 (ABC), were irradiated with single doses over a range of 0-10Gy using photon beams from a Varian 6 MV linear accelerator. Viability and cell cycle were measured at 24 and 48 hrs post-RT using the Muse cell analyzer (EMD Millipore) and FACSAria II (BD Biosciences), respectively. Clonogenic survival in methylcellulose media (0.8%) was determined by imaging after 1 week culture. Results: Overall, RT inhibited cell growth in a dose- and time-dependent manner. At 24 hrs post-RT, SUDHL-6 and WSU-DLCL2 (GCB), as well as SUDHL-2 (ABC) cells showed significantly increased survival as compared to Riva (ABC) at doses of 2Gy (p < 0.05) and higher (p < 0.001). By 48 hrs, SUDHL-6 and SUDHL-2 remained more viable than Riva at doses of 4Gy and higher (p < 0.005), while WSU-DLCL2 became less viable with a pattern similar to Riva. Cell cycle analyses showed the majority of Riva cells in G2/M phase at all doses and time points (71.9%, SD = 0.07), while WSU-DLCL2 and SUDHL-6 cells shifted from G0/G1 to G2/M with increasing doses. Clonogenic survival was higher in WSU-DLCL2 than that of Riva at all radiation doses tested. Conclusions: While the ABC subtype of DLBCL is known for chemoresistance, our data suggest that in some cases, the opposite may be true for radiation therapy–Riva (ABC) showed decreased viability and clonogenic survival compared to GCB subtypes (WSU-DLCL2; SUDHL-6) after RT. In contrast, SUDHL-2 (ABC) behaved like SUDHL-6 and maintained increased viability when compared to Riva and WSU-DLCL2. These findings illustrate the potential impact of disease heterogeneity on the use of RT for patients with different molecular subtypes of DLBCL.

p21-activated kinase-2 (PAK2) is ubiquitously expressed in all mammalian cells and tissues tested so far. It is a unique member of PAK family kinases that can be activated by various stress conditions to induce apoptosis or cytostasis. Although many conditions have been reported to activate PAK2, serum starvation followed by insulin treatment has not been studied. In this study, pre-adipocyte (3T3-L1) sensitive to insulin signaling and important for energy homeostasis was used as the system to address this topic. It was found that serum starvation transiently activated PAK2 activity by about 3-fold within one hour, then returning to the basal level within three hours. Following the activation of PAK2 by serum starvation, insulin treatment resulted in a rapid deactivation of PAK2 through ubiquitination-proteasome mediated protein degradation. AKT1 and PAK2 activities were reversely related, suggesting that AKT1 activation could be a factor to initiate PAK2 degradation. This dynamic change of PAK2 by serum starvation and insulin was found to be correlated with the fluctuation of protein synthesis, a major biological process to influence cell growth rate. Activation of PAK2 by serum starvation was correlated with about 50% inhibition of protein synthesis; subsequent treatment with insulin reversed this inhibition. Down-regulation of PAK2 by siRNA further proved that PAK2 was a causal factor leading to the inhibition of protein synthesis. In conclusion, this study identifies a new pattern of regulation of PAK2 by serum starvation and insulin, suggests an important role of PAK2 in regulating adipocyte function in response to nutrient status and insulin signaling.

Steroid hormone signaling through nuclear receptors plays critical roles in every stage of breast cancer development. Although estrogen receptor (ER) and progesterone receptor (PR) are well studied, due to the high heterogeneity of breast cancer, therapies targeting these nuclear receptors are still not effective enough or generate inconsistent and even controversial results. In this study, we focused on the glucocorticoid (GC)/ glucocorticoid receptor (GR) signaling to investigate how it regulates NFkB activity that leads to differential outcomes in different subtypes of breast cancer. A qRT-PCR array containing a curated NF-kB responsive gene set (84 genes) was utilized in this study to analyze the effect of GC/GR on basal and luminal subtypes of breast cancer cells. It was identified that MDA-MB-231 cells (basal subtype) were more responsive than MCF7 cells (luminal subtype) to GC treatment as the former showed more down- or up-regulated NF-kB target genes. Negative regulation of the NF-kB target genes was more prevalent in both cells, which is consistent with the general rule of trans-repression of NF-kB by GR. However, there were also a number of NF-kB target genes up-regulated by GC in both subtypes of breast cancer cells, suggesting that the trans-activation of NF-kB by GR may also play an important role in breast cancer cell growth and pathological behaviors. Among the differentially expressed genes in both cell types, genes with functions in cell migration and adhesion are highly regulated, such as CXCL1, CCL2, ICAM1, VCAM1, BIRC3 and SNAP25, implying that GC may mainly regulate the invasion and metastasis of breast cancer cells via NF-kB signaling. Biochemical studies identified that GR was activated by GC differently in MDA-MB-231 and MCF7 cells; the interaction between GR and various NF-kB subunits were also different in these cells, thus elucidating part of mechanisms underlying the differential regulation of NF-kB by GC in different subtypes of breast cancer cells. Citation Format: Zenaida Lopez-Dee, Jun Ling. Regulation of NF-kB signaling pathway by glucocorticoid in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1981. doi:10.1158/1538-7445.AM2015-1981

AIM:To evaluate the efficacy of the improved thrombospondin mimetic peptide ABT-898 in a murine model of ulcerative colitis.METHODS:The dextran sodium sulfate (DSS) was used for the induction of colitis in both TSP-1 deficient (TSP-1(-/-)) and wild type (WT) mice during 7 d. While mice were receiving the DSS dissolved in the drinking water, the ABT-898 peptide was dissolved in sterile 5% glucose solution and delivered using mini pumps subcutaneously implanted. Plasma samples were analyzed for interleukin (IL)-6 by ELISA assay and colonic tissues were harvested, fixed and processed for histological evaluation. Immunohistochemistry using antibodies for the detection of CD31 and MECA in endothelial cells was performed. Inflammation was graded in colonic sections and the number of microvessels in each lesion was assessed. Activation of signal transducer and activator of transcription 3 (STAT3) in colonic samples was quantified by immunohistochemistry and Western blotting using antibodies against total STAT3 and phosphorylated STAT3 (pSTAT3) (Ser727).RESULTS:Treatment with ABT-898 considerably diminished the inflammatory response in WT and TSP-1(-/-) mice (P < 0.0001 in both groups vs control). Identification of blood vessels highlighted by CD31/MECA immunohistochemistry, showed significantly reduced vessel counts in colitic lesions of WT and TSP-1(-/-) mice treated with ABT898 (TSP-1(-/-) controls/TSP-1(-/-) treated, P = 0.0002; WT controls/WT treated, P = 0.0005). Consistently, IL-6 was significantly diminished in plasma samples of TSP-1(-/-) and WT treated with the peptide when compared to the control mice (P = 0.0002 and P = 0.0148, respectively). pSTAT3 positive cells were quantified in WT and TSP-1(-/-) treated with ABT-898. A significant decrease in positive cells for pSTAT3 was observed in treated mice (TSP-1(-/-) controls/TSP-1(-/-) treated, P = 0.0089; WT/WT treated, P = 0.0110). These results were confirmed by Western blotting analyses showing lower levels of pSTAT3 in colitic lesions from mice treated with the peptide ABT-898.CONCLUSION:These findings indicate that the new peptide ABT-898 ameliorates inflammation and angiogenesis and might be a therapeutic alternative in IBD and inflammatory diseases.