bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2298: Antibiotic use and breast cancer risk: results from the Sister Study
Authors: Deming-Halverson Sandra L. , Hodgson M. Elizabeth , D'Aloisio Aimee , Shore David , Sandler Dale
Published: July 2017

Background: Regular antibiotic use is hypothesized to influence breast cancer risk by a number of mechanisms including disruption of intestinal microflora which plays a role in the conversion of food-based phytochemicals into bioactive substances suggested to be protective against cancer, and by influences on the immune system and inflammatory response.  Previous results have been mixed, and given the sparse and conflicting data in epidemiologic literature, we analyzed the association between regular antibiotic use and breast cancer risk, overall and by different classes of antibiotics.

Methods: Regular antibiotic use, as characterized by class type and number, duration, and indication for use, was evaluated for its association with breast cancer risk among participants of the NIEHS Sister Study, a prospective cohort of 50,884 US women aged 35-74 without breast cancer who have had a sister diagnosed with breast cancer.

Results: 5,312 (10.4%) of women in the Sister Study reported ever having regular antibiotic use (at least 3 times per week for 3 months in a row or longer) with a median duration of 1.50 years (0.25 – 53.8). 34% of regular uses reported use greater than 2 years. Skin conditions including acne and rosacea were the most common indications for use (6.02%). The hazard ratio (HR) was 1.10 (0.99-1.27) for ever regular antibiotic use and 1.40 (1.16-1.69) for greater than 2 years of regular use. Results are suggestive of an increased risk among regular users who have only ever used one class rather than more than one class of antibiotics. Adjusted hazard ratios were 1.40 (0.99-1.99) for tetracyclines only, 1.45 (0.95-2.20) for penicillins only, 1.42 (0.94-2.15) for macrolides alone. However, there does not appear to be an association between ever regular use of more than one class of antibiotics and breast cancer. Furthermore, there was no association between indications for ever regular use and breast cancer risk, among all women reporting regular use of antibiotics, regardless of class.

Conclusion: Overall, there is a suggestion that ever regular use of a single class of antibiotics, but not use of more than one class, may be associated with increased breast cancer risk. However, given that most individuals reported only a single episode of regular antibiotic use for a duration of less than 2 years; further exploration is warranted to rule out effects of underlying indication for use or explore other possible class specific-confounders.

Citation Format: Sandra L. Deming-Halverson, M. Elizabeth Hodgson, Aimee D'Aloisio, David Shore, Dale Sandler. Antibiotic use and breast cancer risk: results from the Sister Study [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 2298. doi:10.1158/1538-7445.AM2017-2298
Abstract 2305: Residential airborne heavy metal concentrations and breast cancer characteristics
Authors: Kresovich Jacob K. , Erdal Serap , Argos Maria , Chen Hua Yun , Gann Peter H. , Rauscher Garth H.
Published: July 2017

Background: Ambient air metal concentrations have recently been implicated in the etiology of breast cancer. Previous studies have shown airborne concentrations of arsenic and cadmium are associated with the development of estrogen receptor-negative tumors. This study aims to replicate these findings and examine the role of other toxic and essential heavy metals.

Methods: Participants were women who were diagnosed with breast cancer in Chicago between 2005 and 2008. We examined estrogen and progesterone receptor-negative (ER-/PR-) and high-grade tumors as markers of aggressive breast cancer, and estimated 15-year markers of exposure of 11 heavy metals. Exposures were calculated using census tract-level airborne concentrations from the National-scale Air Toxics Assessment and participants’ residential histories. We adjusted all models for socioeconomic status and reproductive factors.

Results: We found that prevalent ER-/PR- tumors were associated with increased residential exposure to airborne concentrations of antimony (Q4 vs Q1: OR= 1.81; 95% CI: 0.95, 3.44; Ptrend= 0.04), cobalt (Q4 vs Q1: OR= 2.37; 95% CI: 1.26, 4.45; Ptrend < 0.01), manganese (Q4 vs Q1: OR= 2.55; 95% CI: 1.24, 5.24; Ptrend= 0.04), and selenium (Q4 vs Q1: OR= 1.85; 95% CI: 1.03, 3.29; Ptrend= 0.05), and also identified marginally significant trends for arsenic (Ptrend= 0.06), chromium (Ptrend= 0.08), lead (Ptrend= 0.08), and mercury (Ptrend= 0.07). We did not identify any overall associations with high-grade tumors, however when stratifying by menopausal status we found that antimony (Q4 vs Q1: OR= 6.97, 95% CI: 1.61-30.19) and arsenic (Q4 vs Q1: OR= 6.97, 95% CI: 1.61-30.19) were associated with prevalent high-grade tumors in premenopausal women only.

Discussion: This study found further support for a role of airborne arsenic concentrations, and novel evidence implicating other airborne estrogen-pathway disrupting metal concentrations, in the development of aggressive breast cancer subtypes. Additionally, this is the first study to implicate heavy metal exposure in the etiology of high-grade tumors. These results suggest that long-term, low-dose exposures to certain heavy metals play a role in the etiology of aggressive breast cancer characteristics. Airborne exposures have the ability to affect large populations and findings from this and similar studies have large public health implications.

Citation Format: Jacob K. Kresovich, Serap Erdal, Maria Argos, Hua Yun Chen, Peter H. Gann, Garth H. Rauscher. Residential airborne heavy metal concentrations and breast cancer characteristics [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 2305. doi:10.1158/1538-7445.AM2017-2305
Abstract 2308: Sigma-2 receptor ligands induce apoptosis and inhibit proliferation in breast cancer cell line mda-mb-231
Authors: Asong Gladys , Amissah Felix , Salako Olufisayo , Poku Rosemary , Ntantie Elizabeth , Nkembo Augustine , Lamango Nazarius , Ablordeppey Seth
Published: July 2017

Introduction: Despite the reduction in mortality from breast cancer achieved in the last 30 years due to more effective targeted therapies, the survival rate for patients with triple negative breast cancer (TNBC) is poor and has virtually plateaued. Due to the absence of estrogen, progesterone, and human epidermal growth factor receptors 2, no targeted treatment options are currently available for TNBC. Moreover, the current standard systemic treatments are known to cause tremendous side effects. Thus, there is an urgent need to identify novel strategies for treating TNBC. Several reports have revealed that sigma-2 receptors are over-expressed in all solid tumors including TNBC, making it a plausible biomarker to explore for TNBC treatment. In this study, we investigated the effects of novel sigma-2 receptor ligands (XYZ-XI-14 and XYZ-VII-69) synthesized in our lab on the viability and survival of the TNBC, MDA-MB-231.

Methods: MDA-MB-231 cells were treated for 48 h with XYZ-XI-14 and XYZ-VII-69, cell viability and proliferation were assessed using resazurin cell titre assay and cell count methods respectively. The effects of the ligands on spheroids formation, cell cycle, and mode of cell death were also investigated.

Results: XYZ-XI-14 and XYZ-VII-69 decreased cell viability of MDA-MB-231 cells in a concentration-dependent manner. The EC50 for XYZ-XI-14 and XYZ-VII-69 were 12 and 13 µM respectively; and at 5 µM, the ligands inhibited cell proliferation, induced apoptosis, and arrested MDA-MB-231 cells at the G0/G1 phase of cell cycle. Additionally, concentrations of the ligand as low as 1 µM prevented the formation of spheroids as evidenced by the lack of compact spheroids, and caused the disintegration of preformed spheroids.

Conclusion: This study indicates that targeting sigma-2 receptors with novel sigma-2 ligands (XYZ-XII-14 and XYZ-VII-69) effectively inhibits TNBC cancer cell growth by inducing apoptosis and cell cycle arrest, thus presenting a unique and effective pathway for treating TNBC. Additionally, the sigma-2 receptor ligands (XYZ-XI-14 and XYZ-VII-69) have the potential to halt tumor growth and prevent tumor relapse, as seen in our 3D culture assays. Thus, the sigma-2 receptor has the potential to be a valuable target for the development of novel agents for the treatment of TNBC.

Citation Format: Gladys Asong, Felix Amissah, Olufisayo Salako, Rosemary Poku, Elizabeth Ntantie, Augustine Nkembo, Nazarius Lamango, Seth Ablordeppey. Sigma-2 receptor ligands induce apoptosis and inhibit proliferation in breast cancer cell line mda-mb-231 [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 2308. doi:10.1158/1538-7445.AM2017-2308
Abstract 2310: Molecular dynamics simulation of permeation pathway of cytochrome C through Bax pore
Authors: Zhang Mingzhen , Zheng Jie , Nussinov Ruth , Ma Buyong
Published: July 2017

How cytochrome C is released from the mitochondria to the cytosol via Bax oligomeric pores, a process which is required for apoptosis, is still a mystery. Based on the residue-residue distances detected experimentally for Bax and its homologous protein (Bak), we recently computationally solve the first atomic model for Bax oligomeric pores at the membranes. Next, we investigate the mechanism at the microsecond time- and nanometer space- scale using coarse-grained replica exchange and all-atom MD simulations. Our free energy landscape depicts a low barrier for the permeation of cytochrome C into the Bax C-terminal mouth, with the pathway proceeding to the inner cavity and exiting via the N-terminal mouth. Release is guided by organized charged/hydrophilic surfaces. The hydrophilicity and negative charge of the pore surface gradually increase along the release pathway from the pore entry to the exit opening. Rather than inert passing of cytochrome C through a rigid pore, the flexible pore may selectively aid the cytochrome C passage. The energy barrier is under 4 kcal/mol. Thus, once the Bax pore is formed in the membrane, the release of cytochrome C may be readily achieved through energy fluctuations. Collectively, our work provides mechanistic insight and atomic detail into the release of cytochrome C through Bax oligomeric pores.

Citation Format: Mingzhen Zhang, Jie Zheng, Ruth Nussinov, Buyong Ma. Molecular dynamics simulation of permeation pathway of cytochrome C through Bax pore [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 2310. doi:10.1158/1538-7445.AM2017-2310
Abstract 2329: TMEM33 induces apoptosis via UPR signaling and autophagy in breast cancer cells
Authors: Hu Rong , Zhang Xiyuan , Hilakivi-Clarke Leena , Kasid Usha , Clarke Robert
Published: July 2017

TMEM33 is a novel transmembrane protein that resides in the endoplasmic reticulum (EnR). It has been shown to activate the PERK and IRE1α branches of the unfolded protein response (UPR). However, the underlying mechanism of action of this EnR resident protein TMEM33 and the cellular functions that it regulates remain largely unknown. In this study, we show that overexpression of TMEM33 induces robust cell death in breast cancer cells. Inhibition of the PERK/eIF2alpha pathway with eIF2alpha inhibitor ISRIB blocks the TMEM33 induced cell death. TMEM33 overexpression strongly activates UPR associated pro-death JNK-p53 signaling. We also observed a significant inhibition of the downstream survivin, which blocks cell death activation by binding to caspases and inhibiting their activation. We further show that the blockage of JNK activation with either an inhibitor or overexpression of survivin, protects cells against TMEM33 induced apoptosis. In addition, we show that TMEM33 overexpression induces autophagy in breast cancer cells. Inhibition of autophagy with using either the inhibitor chloroquine or knockdown of the Atg5 gene, further sensitizes breast cancer cells to the effects of TMEM33 overexpression. Cell death induced by TMEM33 is also decreased by overexpression of the autophagy gene Beclin 1. The findings in this study demonstrate that the novel EnR resident protein TMEM33 induces cell death by activating IRE1α-JNK-p53-survivin signaling in breast cancer cells. Concurrently, autophagy is also activated by TMEM33, and functions as a pro-survival mechanism. Cell fate reflects the balance between the pro-death and pro-survival activities as regulated by TMEM33.

Citation Format: Rong Hu, Xiyuan Zhang, Leena Hilakivi-Clarke, Usha Kasid, Robert Clarke. TMEM33 induces apoptosis via UPR signaling and autophagy in breast cancer cells [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 2329. doi:10.1158/1538-7445.AM2017-2329
Abstract 2332: Integral modulation of nuclear factor-kappa B activation by C/EBPβ and the endoplasmic reticulum stress sensor PERK to mediate estrogen-induced apoptosis in estrogen-deprived breast cancer cells
Authors: Fan Ping , Tyagi Amit K. , Agboke Fadeke A. , Pokharel Niranjana , Jordan V. Craig
Published: July 2017

Stress responses are critical for estrogen (E2) to induce apoptosis in E2-deprived breast cancer cells. Nuclear factor-kappa B (NF-κB) is well known as a therapeutic target to prevent stress responses in chronic inflammatory diseases including cancer. However, whether E2 activates NF-κB to participate in stress-associated apoptosis in E2-deprived breast cancer cells is unclear. We demonstrated that E2 differentially modulates NF-κB activity in E2-deprived breast cancer cells according to the treatment time. Because E2 initially has significant potential to down modulate the NF-κB activation, it completely suppresses the tumor necrosis factor alpha (TNFα)-induced NF-κB activation. We found that E2 preferentially and constantly enhances the expression of transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) which is responsible for suppression of NF-κB activation by E2 in MCF-7:5C cells. The mTOR signaling pathway promotes repression of NF-κB by C/EBPβ which is confirmed by the evidence that inhibition of mTOR is synergistic with E2 to upregulate NF-κB-dependent genes, such as TNFα. Interestingly, NF-κB p65 activity is upregulated when E2-treatment is administered for 48 hours, leading to induction of TNFα. Blocking the nuclear translocation of NF-κB completely prevents E2 from induction of TNFα and apoptosis. Importantly, protein kinase RNA-like endoplasmic reticulum kinase (PERK), a stress sensor of unfolded protein response, is activated by E2 and plays an essential role in increasing NF-κB p65 DNA binding through the activation of STAT3, independently of canonical IκBα signal pathway. Thus, inhibition of PERK kinase activity completely blocks nuclear activation of NF-κB and NF-κB-dependent induction of TNFα, thereby preventing E2-induced apoptosis. All of these findings illustrate a crucial role for the novel PERK/NF-κB/TNFα axis in E2-induced apoptosis which is integrally modulated by the stress responsive transcription factor C/EBPβ and endoplasmic reticulum stress. This study provides an important rationale for exercising caution in clinical trials when considering targeting PERK or NF-κB following the development of acquired resistance to aromatase inhibitors whereas mTOR may be a target to enhance the therapeutic effects of E2 in antihormone resistant breast cancer.

Citation Format: Ping Fan, Amit K. Tyagi, Fadeke A. Agboke, Niranjana Pokharel, V. Craig Jordan. Integral modulation of nuclear factor-kappa B activation by C/EBPβ and the endoplasmic reticulum stress sensor PERK to mediate estrogen-induced apoptosis in estrogen-deprived breast cancer cells [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 2332. doi:10.1158/1538-7445.AM2017-2332
Abstract 2342: TNFAIP8 promotes prostate cancer cell survival by modulating autophagy
Authors: Niture Suresh , Ramalinga Malathi , Kedir Habib , Kumar Deepak
Published: July 2017

TNF-α inducible protein (TNFAIP8) is an antiapoptotic protein with roles in tumor cell growth and survival. Mechanisms of cell survival by TNFAIP8 remains elusive. In the current study, we investigated the role and molecular mechanism related to TNFAIP8 in the modulation of cell/cycle, autophagy and drug resistance/cell survival in prostate cancer cells. Microarray data from PC3 prostate cancer cells ectopically expressing TNFAIP8 demonstrated modulation of cell cycle related genes such as CDKs, CDCs and PCNA. Immunoblotting data from overexpression of TNFAIP8 in PC3 cells shows upregulation of cell cycle proteins cyclin A, cyclin B1, Myt1 and Chk1. However, no change in cell cycle was found. Phosphorylation of Histone-S10, CDC2-Tyr15 and Wee1-S642] was also observed. Autophagy plays an important role in tumor cell survival. We evaluated the effects of TNFAIP8 in modulating autophagy. Overexpression of TNFAIP8 leads to induction of autophagy. TNFAIP8 positively modulates the expression/stabilization of autophagy markers and effectors such as LC3β I/II, Beclin1, 4EBP1, oncogene p62 and SIRT1. Knockdown of TNFAIP8 inhibits autophagy induced by nutrient starvation in PC3 prostate and MCF7 breast cancer cells. We also evaluated cell growth and survival in PC3 cells. We demonstrate that TNFAIP8 promotes cell growth and proliferation in PC3, LNCaP and C4-2 prostate cancer cells. Furthermore, we have found that TNFAIP8 increase resistance against anticancer drugs docetaxel and doxorubicin. These data collectively suggest that by the creation of cellular autophagy events TNFAIP8 promotes cell survival and drug resistance in prostate cancer cells.

Citation Format: Suresh Niture, Malathi Ramalinga, Habib Kedir, Deepak Kumar. TNFAIP8 promotes prostate cancer cell survival by modulating autophagy [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 2342. doi:10.1158/1538-7445.AM2017-2342
Abstract 2345: CDK4 inhibition enhances anti-melanoma effects of BRAF/MEK inhibition
Authors: Zhang Yanping , Hsueh Eddy C.
Published: July 2017

Introduction: Targeted therapy against BRAF-mutated melanoma has shown clinical efficacy. However, resistance to therapy often occurs. CDK4 (INK4)-retinoblastoma (RB) pathway controls cell cycle progression by regulating the G1-S checkpoint. Defect in the p16INK4A: cycling D-CDK4/6: RB pathway is also detected in melanoma. We study the effect of combining CDK4-RB pathway inhibition with BRAF/MEK inhibition as a possible strategy to overcome resistance to therapy.

Methods: Three BRAF wild-type and three BRAF-mutated melanoma cell lines were selected. CDK4/6 inhibitor (PD0332991; PD), BRAF inhibitor (PLX4032; PLX), MEK inhibitor (AZD6244; AZD), and PI3K/AKT inhibitor (XL765; XL) were used alone or in combination. Cell proliferation assay was performed using Cell Titer Blue assay. Cell migration assay was performed by artificial wounding of melanoma cell monolayer and observing migration of cells into the wound up to 48 hours. Western blotting was performed for expression of pERK/ERK, pRB/RB, Cyclin D1, CDK4, and GAPDH. Data were presented as means ± SD for triplicate experiments. For comparison between groups, the student’s t test was used and p<0.05 was considered to be statically significant.

Results: Variable inhibition of cell proliferation and migration was observed in a dose- and time-dependent fashion. Enhanced inhibition of melanoma proliferation and migration was observed with PD+AZD and PD+PLX combination compared with single agent AZD or PLX. Differential enhancement was observed in BRAF wild-type cell lines with PD+AZD and in BRAF mutated cell lines with PD+PLX combination. Minimal change in tumor proliferation and migration inhibition was noted with PD+XL combination compared with PD or PLX alone. No correlation was observed between expression of CDK4 and cyclin D1 and sensitivity to singe agent or the combination. Inhibition of phosphorylated RB1 and ERK expression was observed with exposure to single agent PD, PLX, XL, AZD. Enhancement of pRB1 and pERK inhibition was noted with PD+PLX and PD+AZD combination but not PD+XL.

Conclusion: CDK4 inhibition can enhance anti-proliferation and migration effects of BRAF/ MEK inhibition in melanoma cells. The mechanism is via augmented down-regulation of pRB expression.

Citation Format: Yanping Zhang, Eddy C. Hsueh. CDK4 inhibition enhances anti-melanoma effects of BRAF/MEK inhibition [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 2345. doi:10.1158/1538-7445.AM2017-2345
Abstract 2352: Effect of inhibition of cell cycle versus transcription cyclin-dependent kinases (CDKs) in ovarian cancer cells
Authors: Chilà Rosaria , Panini Nicolò , Erba Eugenio , Damia Giovanna , Broggini Massimo
Published: July 2017

Background. Cell cycle is regulated by cyclin-dependent kinases (CDKs) activity, whose deregulation can lead to uncontrolled proliferation and cancer. Other CDKs are engaged in the regulation of transcription and post-transcriptional mRNA processing through the phosphorylation of the C-terminal domain of RNA polymerase II, such as CDK9 and CDK12. Inhibitors of cell cycle CDKs have been developed as anticancer agents and some of them are under clinical validation (e.g. palbociclib). While recent data would suggest that inhibition of CDK9 is feasible and has antitumor effect, the data on the therapeutic role of CDK12 inhibition are very scanty.

Methods. Ovarian cancer cell lines were maintained in RPMI medium supplemented with 5% glutamine and 10% FBS. Cells were treated with different drug concentrations and after 72 hours cell survival was evaluated by MTS assay (Promega). IC50 values were calculated by interpolation method. Cell cycle analysis and apoptosis were performed with standard flow cytometric methods. A2780 and SKOV3 ovarian cancer cell lines knocked out for CDK12 were generated with CRISPR/CAS9 genome editing tool.

Results. The cytotoxicity of palbociclib (a CDK4/6 inhibitor) and LDC000067 (a CDK9 inhibitor) was tested in a panel of ovarian cancer cell lines (A2780, SKOV3, OVCAR3, OVCAR5, OVCAR8, OVCA432, OVCA433, IGROV1, EFO27). Sensitivity of cells was similar for palbociclib and LDC000067, ranging from 10 to 33 and from 8 to 60 μM, respectively. A preferential G1 block was observed with palbociclib, while LDC000067 caused a S-G2 block. A higher induction of apoptosis was observed after LDC000067 than after palbociclib treatment in both A2780 and SKOV3. The palbociclib-induced G1 block was associated with decreased Rb phosphorylation, while no modulation of the Ser2 in the carboxyterminal domain of RNA polymerase II was observed after LDC000067 treatment. We generated CDK12 knocked out cells transfecting CRISPR/CAS9 engineered plasmid in both A2780 and SKOV3 ovarian cancer lines. The biological and pharmacological characterization of these clones is under study.

Conclusions. Palbociclib and LDC000067 showed a dose dependent cytotoxic effect in the panel of ovarian cancer cell lines tested and were active in the μM range. Preliminary data of treatment induced cell cycle perturbation and apoptosis suggest that the two drugs behave in a different manner and have distinct molecular effects on cells.

Citation Format: Rosaria Chilà, Nicolò Panini, Eugenio Erba, Giovanna Damia, Massimo Broggini. Effect of inhibition of cell cycle versus transcription cyclin-dependent kinases (CDKs) in ovarian cancer cells [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 2352. doi:10.1158/1538-7445.AM2017-2352
Abstract 2353: CDKI-15, a novel and highly selective CDK4/6 inhibitor: discovery, in vitro and in vivo anticancer efficacy
Authors: Tadesse Solomon , Bantie Laychiluh , Tomusange Khamis , Islam Saiful , Rahaman Muhammed H. , Noll Benjamin , Lam Frankie , Yu Mingfeng , Wang Shudong
Published: July 2017

Cyclin D dependent kinases CDK4 & CDK6 are crucial regulators of the G1 to S phase transition of the cell cycle. The facts that myriad cancer types show aberrance in INK4-CDK4/6-cyclin D-Rb-E2F pathway, & the rapidly emerging positive clinical data of pharmacological inhibitors have validated CDK4/6 as anticancer drug targets. As the first commercialized CDK inhibitor, palbociclib in combination with letrozole or fulvestrant has received regulatory approval for the treatment of breast cancer. This represents an important scientific advance in the field. However, the limited structural diversity & undesired side effects due to broader kinase interactions of existing inhibitors mean that the hunt for new & highly selective CDK4/6 inhibitor drug candidates continues. Using our advanced medicinal chemistry, targeted biochemical & cell-based assays, & animal pharmacology, we synthesized & evaluated a novel series of inhibitors. Many compounds were highly potent & selective against CDK4/6, & exhibited low nanomolar potency against a range of human cancer cell lines. Notably, inhibition of CDK4D1 by compound CDKI-15 (Ki = 4 nM) was over 3 orders of magnitude greater than CDK1B, CDK2A, CDK7H & CDK9T1. Interrogation of a panel of 369 protein kinases revealed CDKI-15 to be highly selective for CDK4/6 with only 3 other kinases inhibited potently. CDKI-15 reduced the level of Rb phosphorylation & induced G1 cell cycle arrest, confirming cellular inhibition of CDK4/6 in cancer cells. Moreover, CDKI-15 possesses superior pharmacokinetic profile with oral bioavailability of 100% in mice. Treatment of nude BALB/c mice bearing human MV4-11 acute myeloid leukemia cells with CDKI-15 at daily dose of 100 mg/kg by oral administration resulted in a robust inhibition of tumor growth compared to vehicle treated animals (T/C = 30%, p < 0.00001). Strikingly, CDKI-15 caused a complete & sustained tumor regression in one-third of the animals. No detectable toxicity was observed in the animals during & post treatment. Taken together, our data provide a rationale for CDKI-15 to be developed towards clinic for cancer therapy.

Citation Format: Solomon Tadesse, Laychiluh Bantie, Khamis Tomusange, Saiful Islam, Muhammed H. Rahaman, Benjamin Noll, Frankie Lam, Mingfeng Yu, Shudong Wang. CDKI-15, a novel and highly selective CDK4/6 inhibitor: discovery, in vitro and in vivo anticancer efficacy [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 2353. doi:10.1158/1538-7445.AM2017-2353
Abstract 2354: Identification of CDK1 as an aspirin and salicylic acid binding protein: a potential role in chemoprevention
Authors: Gunaje Jayarama B. , Kumar D.Ramesh , Kesharwani Siddharth , Callegari Eduardo , Tummala Hemachand , Dachineni Rakesh
Published: July 2017

Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin for chemoprevention; however, the mechanisms leading to its anti-cancer effects are still being elucidated. We hypothesized that aspirin’s ability to exert chemopreventive effects may involve altering the levels and activity of cell cycle regulatory proteins. In the present study, using HT-29 colon cancer cells and other cancer cells, we demonstrated that both aspirin and its primary metabolite, salicylic acid, downregulated the protein and mRNA levels of cyclin B1 and cyclin dependent kinase-1 (CDK1). Lactacystin, a 26S proteosomal inhibitor, prevented aspirin and salicylic acid mediated degradation of cyclin B1, but not CDK1. Decrease in protein levels of cyclin B1/CDK1 was correlated with a corresponding decrease in CDK1 kinase activity. Molecular docking studies showed that aspirin and salicylic acid independently can dock on CDK1 through interactions with Leucine 83. Incubation of recombinant CDK1 with aspirin resulted in acetylation at lysine residues, this was also observed in cell culture experiments. Pre-incubation of CDK1 with salicylic acid dose dependently prevented aspirin’s ability to acetylate CDK1 in purified preparations confirming the data obtained from molecular docking studies. Our results show that CDK1 is a salicylic acid binding protein (SABP) and the chemopreventive actions of aspirin may involve modulation of levels and activity of cyclin B1 and CDK1.

Citation Format: Jayarama B. Gunaje, D.Ramesh Kumar, Siddharth Kesharwani, Eduardo Callegari, Hemachand Tummala, Rakesh Dachineni. Identification of CDK1 as an aspirin and salicylic acid binding protein: a potential role in chemoprevention [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 2354. doi:10.1158/1538-7445.AM2017-2354
Abstract 2361: Pim kinase inhibition alters mRNA splicing in AML cell lines
Authors: Joglekar Tejashree A. , Li Xiang , Bieberich Charles J.
Published: July 2017

Oncogenic kinase activity is a common feature of nearly all cancers and kinases are major targets for therapeutic intervention. Pim kinases are deregulated in hematopoietic cancers including Acute Myeloid Leukemia (AML), as well as prostate cancer. While cancer cells can become dependent on Pim activity to sustain proliferation, in normal adult tissues, Pim kinase activity appears to be dispensable. These features make Pim kinases an attractive target for cancer therapy, however, their physiological roles have not been fully characterized. Using the reverse in-gel kinase assay (RIKA), we identified a battery of novel Pim substrates that are involved in mRNA splicing regulation. We hypothesized that Pim family kinases regulate mRNA splicing through phosphorylation of splicing factors. Microarray analysis revealed more than 10,000 splicing changes in AML cells treated with the highly selective small molecule Pim kinase inhibitor AZD1208. Using RT-PCR analysis, multiple AZD1208-induced splicing changes were validated. To discern the mechanisms whereby Pim kinases regulate splicing, we determined whether Pim inhibition alters phosphorylation of serine/arginine-rich (SR) proteins. Inhibition of serine arginine protein kinase (SRPK) activity using a small molecular inhibitor (SRPIN340) reduced SR protein phosphorylation, whereas Pim kinase inhibition did not, suggesting that Pim kinases regulate splicing through an SRPK independent pathway. Comparison of splicing changes in AZD1208 and SRPIN340 treated AML cells demonstrated distinct patterns, providing further evidence of SRPK-independent splicing regulation by Pim kinases. Biomarkers of kinase inhibitor efficacy are critical components of clinical trials. These data suggest that changes in mRNA splicing may serve as a biomarker for assessing patient responsiveness to Pim inhibition therapy.

Citation Format: Tejashree A. Joglekar, Xiang Li, Charles J. Bieberich. Pim kinase inhibition alters mRNA splicing in AML cell lines [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 2361. doi:10.1158/1538-7445.AM2017-2361
Abstract 2365: Dual inhibition of the PI3k/Akt and MEK5/ERK5 pathways in tamoxifen resistant breast cancer
Authors: Wright Thomas D. , Hasan Mahmud , Witt-Enderby Paula , Cavanaugh Jane
Published: July 2017

Approximately 70% of breast cancers are considered estrogen receptor alpha (ER) and/or progesterone receptor (PR) positive, and their growth is primarily driven by the hormone estrogen. Initially these cancers are responsive to endocrine therapies such as tamoxifen. However, over time patients become resistant to conventional endocrine therapy and treatment becomes more difficult. Tamoxifen resistance occurs due to several mechanisms: loss of ER expression, gain of function mutations, pharmacological tolerance, alterations of co-activators, and ligand independent activation of ER by various kinase cascades. Aberrations in the Phosphoinositide-3-kinase (PI3K) and Mitogen Activated Protein Kinase (MAPK) pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. The crosstalk between PI3K/Akt and MEK1/2/ERK1/2 has been characterized in several systems. However, new evidence suggests that MEK5/ERK5, a member of the MAPK family, is a key component in the proliferation and survival of therapy resistant cancers. MEK5/ERK5 has been shown to promote ER alpha driven transcription in ER+ breast cancers and actin reorganization and metastasis in ER- breast cancers. Furthermore, MEK5/ERK5 promotes hormone independent tumorigenesis in breast cancer. Our lab has previously investigated these pathways in hormone independent breast cancers, TNBC (triple negative breast cancer). Our previous results indicate that combinations of PI3k/Akt and MEK5/ERK5 blockade are promising because they inhibit both the pro-proliferative and pro-metastatic pathways in TNBC. Additionally, PI3k/Akt and MEK5/ERK5 inhibition was shown synergistically reduce viability in a diverse panel of cell lines (TNBC, ER+, and Triple Positive). Based on these initial results we hypothesize that PI3k/Akt and MEK5/ERK5 inhibition decreases viability via a hormone independent mechanism: perhaps by reducing the cytosolic sequestration of Bad. In this study we investigate the utility PI3k/Akt and MEK5/ERK5 inhibition in a MCF-7 TamR cell line. The goal of our study is to elucidate the roles of PI3k/Akt/mTOR and MEK5/ERK5 in endocrine resistant breast cancer and broaden the scope of a dual inhibition strategy.

Citation Format: Thomas D. Wright, Mahmud Hasan, Paula Witt-Enderby, Jane Cavanaugh. Dual inhibition of the PI3k/Akt and MEK5/ERK5 pathways in tamoxifen resistant breast cancer [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 2365. doi:10.1158/1538-7445.AM2017-2365
Abstract 2368: Differential role of Pik3caH1047R and Pten-/- mutations in thyroid cancer development
Authors: Ranieri Michela , Cristofano Antonio Di
Published: July 2017

Anaplastic thyroid cancer (ATC) is one of the most lethal malignancies, with a median survival of less than 6 months from time of diagnosis.

Molecular changes that characterize ATC involve most often p53 loss or inactivation (up to 80%), activation of the PI3K cascade through PTEN loss (12%) and PIK3CA activating mutations (20%) or gene amplification (30-60%), RAS family members activation (6-50%), and BRAF activation (25-50%).

Studies performed in endometrial and breast cancer suggest that although PI3K activating mutations and PTEN loss of function both enhance PI3K signaling, these mutations are not equivalent.

We have tested whether Pik3caH1047R and Pten-/- mutations have the same impact on thyroid cancer development and if these two mutations differ in their ability to cooperate with additional genetic alterations, such as p53 deletion and the KrasG12D activating mutation.

We have generated a series of mouse models and cell lines carrying thyroid-restricted p53-/-, KrasG12D, Pten-/-, and Pik3caH1047R alleles.

Analysis of single and compound mutants has revealed important and striking differences between Pten and PI3K abilities to control the PI3K signaling pathway.

Tumor development analysis in Pten-/- and Pik3caH1047R mice has revealed that Pik3ca activation is less potent in inducing thyroid hyperplasia and subsequent transformation than Pten loss, even though the two mutations appear to have similar ability to induce AKT activation. This differential activity correlates with lower levels of PIP3 found in the thyroids of Pik3caH1047R mice, compared to that of Pten-/- mice.

We also observed that while Pten loss synergizes with Trp53 loss to induce anaplastic thyroid cancer, activation of Pik3ca leads to the development of a smaller and well differentiated tumors.

On the other hand, Pten loss and Pik3ca activation are equivalent in inducing poorly differentiated tumors in the presence of a Kras activating mutation.

However, KrasG12D, Pik3caH1047R cell lines are more sensitive to PI3K, MEK and combined PI3K/MEK inhibition than KrasG12D, Pten-/- cell lines.

Accordingly, tumors developing in mice carrying the KrasG12D, Pik3caH1047R alleles are more sensitive than KrasG12D, Pten-/- mice to a dual PI3K/mTor inhibitor as well as a MEK inhibitor.

All together these results suggest that Pten loss has a stronger impact than Pik3ca activation to thyroid cancer development and resistance to small molecule inhibitors.

Citation Format: Michela Ranieri, Antonio Di Cristofano. Differential role of Pik3caH1047R and Pten-/- mutations in thyroid cancer development [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 2368. doi:10.1158/1538-7445.AM2017-2368
Abstract 2370: Depletion of p21-activated kinase 4, PAK4, inhibits cellular proliferation, motility and clonogenicity in oral squamous cell carcinoma
Authors: Chung Felicia F. , Tan Si-Hoey , Raja Vijay J. , Ng Pei Yuen , Paterson Ian C. , Tan Chye Ling , Leong Chee-Onn
Published: July 2017

Oral squamous cell carcinoma (OSCC) accounts for 90% of all malignancies in the oral cavity, and is often associated with low survival and poor prognosis. While considerable progress in OSCC treatment has been made, options for targeted therapy remain limited. Thus, it is imperative that new druggable targets are identified and characterized. p21-activated kinase 4 (PAK4) is a member of the PAK family of serine/threonine kinases, which are known modulators of a diverse range of oncogenic pathways including cell proliferation, escape from apoptosis, and anchorage-independent growth. Previous reports have indicated that PAK4 is overexpressed in a subset of invasive OSCC, and is highly associated with poor prognosis. However, the specific role of PAK4 in OSCC has yet to be elucidated. Here, we show that RNAi-mediated depletion of PAK4 conferred reduced clonogenicity, cell motility, and cell proliferation in OSCC cell lines in a cell line-dependent manner. Global proteomic profiling of PAK4-depleted H103 and H357 cells was conducted to identify the molecules and signaling pathways responsive to PAK4 depletion. In addition to reported targets of PAK4 such as CFL1, ACTB, and TUBB6, we observed that p16INK4a, pre-mRNA splicing factors (SRSF2, SRSF5, SRSF6, SLU7), and proteasome activator subunits (PSME1-4) were dysregulated as a result of PAK4 depletion. These results indicate that PAK4 plays a role in promoting OSCC progression by regulating multiple signaling pathways. Of note, there have yet to be any reports describing interactions between PAK4 signaling and mRNA splicing or proteasome activation. Further investigation is thus warranted to further delineate the potential role of PAK4 in regulating these targets.

Citation Format: Felicia F. Chung, Si-Hoey Tan, Vijay J. Raja, Pei Yuen Ng, Ian C. Paterson, Chye Ling Tan, Chee-Onn Leong. Depletion of p21-activated kinase 4, PAK4, inhibits cellular proliferation, motility and clonogenicity in oral squamous cell carcinoma [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 2370. doi:10.1158/1538-7445.AM2017-2370
Abstract 2372: Xkr5 negatively regulates KIT/D816V signaling
Authors: Sun Jianmin , Thingholm Tine , Li Tianfeng , Kazi Julhash U. , Zhao Hui , Rönnstrand Lars
Published: July 2017

D816V is the most often occurred mutation of type III receptor tyrosine kinase KIT in mastocytosis and CBF-AML. In attempting to identify specific downstream signaling pathways of KIT/D816V, we found that KIT/D816V but not wild-type KIT can bind to Xkr5 that was not studied previously. In addition to the association with KIT/D816V, Xkr5 is phosphorylated at Tyr 369, Tyr487 and Tyr 543 by KIT/D816V, and the phosphorylation is mediated by only mutations of Asp 816 in KIT but not by other KIT mutations that occurred in gastrointestinal stromal tumors and melanoma. Furthermore, the tyrosine phosphorylation of Xkr5 is not dependent on Src family kinases that play a crucial role in the activation of wild-type KIT. In KIT/D816V expressing mast cells, phosphorylation of Xkr5 inhibits KIT/D816V downstream signaling molecules Akt, Erk and p38. As a consequence, cell proliferation and colony formation are inhibited by the tyrosine phosphorylation of Xkr5 as well, indicating that Xkr5 is a negative regulator of KIT/D816V signaling.

Citation Format: Jianmin Sun, Tine Thingholm, Tianfeng Li, Julhash U. Kazi, Hui Zhao, Lars Rönnstrand. Xkr5 negatively regulates KIT/D816V signaling [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 2372. doi:10.1158/1538-7445.AM2017-2372
Abstract 2383: Nicotinamide N-methyltransferase inhibits Protein Phosphatase 2A activity by promoting Y307 phosphorylation
Authors: Jacob John R. , Chakravarti Arnab , Palanichamy Kamalakannan
Published: July 2017

Recent evidence indicates that suppression of protein phosphatase 2A (PP2A) activity plays an essential role in malignant transformation and cancer cell maintenance. Despite the importance of PP2A as a tumor suppressor in cancer, the mechanisms by which other enzymes regulate PP2A remain largely unknown. We recently identified Nicotinamide N-methyltransferase (NNMT) as a novel regulator of PP2A activity in glioblastoma (GBM). NNMT is a cytosolic N-methyl transferase which utilizes methyl groups from S-Adenosyl methionine (SAM), forming the by-product S-Adenosyl Homocysteine (SAH). PP2A becomes activated following the trimer formation of the three PP2A subunits, which requires the methylation of the catalytic subunit of PP2A (PP2A C) at L309.In cancer, NNMT has been reported to decrease the cellular methylation potential (SAM:SAH) and correspond with decreased PP2A activity. It was hypothesized that the decrease in methylation potential accounts for the decrease in PP2A activity by kinetically disfavoring the methylation of PP2A C. Interestingly, when we overexpressed NNMT in GBM cell lines and used LC-MS/MS (LC-MS Quadrupole Time-of-Flight and LC-MS Triple Quad) to monitor the methylation potential, we discovered that overexpression of NNMT increased methylation potential, however, still lead to a decrease in PP2A activity. As a result, we hypothesized that NNMT suppresses PP2A activity in a methylation-independent manner. In an effort to identify an alternative mechanism by which NNMT decreases PP2A activity, we found that NNMT expression was required for the phosphorylation of PP2A C at residue T307, which inhibits PP2A trimer formation and activation. Further, we found that NNMT expression leads to the constitutive activation of the PP2A target serine/threonine kinases (STKs): Akt, MAPK, and SAPK/JNK. NNMT silencing resulted in the deactivation of these STKs and radiation-induced cellular stress was not sufficient to rescue their activation. This suggests that NNMT-induced PP2A suppression is essential for the activation of these kinases. Overall, this study demonstrates a novel mechanism independent of methylation by which NNMT represses the activity of tumor suppressor PP2A.

Citation Format: John R. Jacob, Arnab Chakravarti, Kamalakannan Palanichamy. Nicotinamide N-methyltransferase inhibits Protein Phosphatase 2A activity by promoting Y307 phosphorylation [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 2383. doi:10.1158/1538-7445.AM2017-2383
Abstract 2385: Systematic characterization of kinase inhibitors reveals heterogeneity in responses by class and cell line
Authors: Mills Caitlin E. , Hafner Marc , Juric Dejan , Sorger Peter K.
Published: July 2017

Several publications have addressed concerns surrounding drug response screens by pointing out sources of variability and by presenting recommendations for better experimental methods and more robust analytical approaches. In the presented profiling effort, we integrated the latest advances in drug response measurement and focused on data diversity and quality rather than on breadth. We selected 32 breast cancer cell lines with a strong bias towards triple negative lines as well as 4 cell lines established from relevant patient-derived xenografts. We evaluated a panel of clinically relevant kinase inhibitors using a microscopy-based drug response assay to measure drug potency, and to quantify drug efficacy in terms of growth inhibition (GR metrics) and cell death. The use of the GR metrics to quantify drug sensitivity enabled us to identify and study differences between cytostatic and cytotoxic responses. This systematic dose response dataset is complemented by measurements of baseline mRNA expression levels by RNAseq and endogenous protein levels by shotgun mass spectrometry across all cell lines. The completeness and controlled conditions under which these data sets were collected provide confidence in their integration. The baseline RNA and protein expression levels were used to build predictors of the measured drug responses with the goal of identifying the biological processes responsible for the differences in sensitivity across drugs and cell lines. Differences in the phenotypic responses of cell lines to kinase inhibitors with the same nominal targets have been investigated, and associated with variable inhibitor polypharmacology.

Citation Format: Caitlin E. Mills, Marc Hafner, Dejan Juric, Peter K. Sorger. Systematic characterization of kinase inhibitors reveals heterogeneity in responses by class and cell line [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 2385. doi:10.1158/1538-7445.AM2017-2385