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Supplemental Figure 1. Notch4 mRNA expression correlates with Notch ligands and downstream effectors; Supplemental Figure 2. A schematic outline of experimental procedures; Supplemental Figure 3. A single Notch+ cell has the capacity to generate tumors that represents the initial heterogeneity; Supplemental Figure 4. Evaluation of tumor growth following the treatment of animals with GSI, Docetaxel or combination treatments.

APOBEC and AICDA expression after exposure to gefitinb.

Uracil-DNA glycosylase (UDG) hydrolysis assay.

AICDA CRISPR1 and CRISPR2 mutations.

Patients' clinical data.

Sensitivity of T790M ddPCR assay.

AICDA and RELB expression after TKI exposure was inhibited upon pretreatment with BAY11-7802.

Supplementary Figure S1. BRCA1 mutation confirmation and RAD51 gene expression in SUM149 cells. Supplementary Figure S2. RAD51 foci is associated with CSC population of BRCA1-mutant TNBC cells and effect of long-term PARP inhibition on RAD51. Supplementary Figure S3: Knockdown of RAD51 prevents foci formation in SUM149 and SUM159 cells. Supplementary Figure S4. The effect of RAD51 KD on TNBC viable cell count, CSCs, and cell cycle. Supplementary Figure S5. RAD51 KD sensitizes TNBC CSCs to PARPi. Supplementary Figure S6. Efficiency of RAD51 shRNA knockdown with additional shRNA vectors. Supplementary Figure S7. Knocking down RAD51 with two additional shRNA vectors also sensitizes CSCs to PARPi. Supplementary Figure S8. "Physiologic" RAD51 knockdown decreases cancer stem cells in SUM149 cells.

Almost all patients with EGFR-driven lung cancer who are treated with EGFR tyrosine kinase inhibitors (TKI) develop resistance to treatment. A single base (c.2369C>T) transition mutation, EGFR T790M, is the most frequent resistance event after first-generation exposure to EGFR TKIs. Whether T790M mutation is acquired or is selected from a preexisting clone has been a matter of significant debate. In this study, we show that treatment with EGFR TKIs leads to activation of the NFκB pathway, which in turn induces expression of activation-induced cytidine deaminase (AICDA). In turn, AICDA causes deamination of 5-methylcytosine to thymine at position c.2369 to generate the T790M mutation. Pharmacologic inhibition of the NFκB pathway or knockout of AICDA decreased the frequency or prevented the development of T790M mutation, respectively. In addition, patients treated with first-line EGFR TKI displayed increased expression of AICDA and detection of the T790M mutation upon progression. These results identify the mechanism of T790M acquisition and present an opportunity to target the process to delay or prevent it.Significance:These findings identify the mechanism behind acquisition of a common resistance mutation to TKI treatment in lung cancer.

Supplementary figures 1-8 legends

Deamination of Cytosine or 5-methylcytosine to Uracil or Thymine respectively.

Elevated levels of the proinflammatory cytokine IL6 are associated with poor survival outcomes in many cancers. Antibodies targeting IL6 and its receptor have been developed for chronic inflammatory disease, but they have not yet been shown to clearly benefit cancer patients, possibly due to antibody potency or the settings in which they have been tested. In this study, we describe the development of a novel high-affinity anti-IL6 antibody, MEDI5117, which features an extended half-life and potent inhibitory effects on IL6 biologic activity. MEDI5117 inhibited IL6-mediated activation of STAT3, suppressing the growth of several tumor types driven by IL6 autocrine signaling. In the same models, MEDI5117 displayed superior preclinical activity relative to a previously developed anti-IL6 antibody. Consistent with roles for IL6 in promoting tumor angiogenesis, we found that MEDI5117 inhibited the growth of endothelial cells, which can produce IL6 and support tumorigenesis. Notably, in tumor xenograft assays in mice, we documented the ability of MEDI5117 to enhance the antitumor activities of chemotherapy or gefitinib in combination treatment regimens. MEDI5117 also displayed robust activity on its own against trastuzumab-resistant HER2+ tumor cells by targeting the CD44+CD24− cancer stem cell population. Collectively, our findings extend the evidence of important pleiotropic roles of IL6 in tumorigenesis and drug resistance, and offer a preclinical proof of concept for the use of IL6 antibodies in combination regimens to heighten therapeutic responses and overcome drug resistance. Cancer Res; 76(2); 480–90. ©2016 AACR.

PDF file - 491K, Supplementary Figure 3. Effect of trastazumab on stem cell marker expression. Supplementary Figure 2. Trastuzumab inhibits tertiary tumorsphere formation in luminal cell lines. Supplementary Figure 1. Representative FACS analysis demonstrating an overlap between HER2 expression and ALDH activity. Supplementary Figure 4. Trastuzumab treatment or HER2 gene knockdown reduces tumorsphere formation of MCF7 cells. Supplementary Figure 5. Rank mediated upreglation of HER2 expression in MCF7 cells. Supplementary Figure 6. Elevated HER2 expression in bone metastasis compared to matched primary luminal tumors is not due to HER2 gene amplification. Supplemental Figure 7. No significant differences in HER2 protein expression were found in matched primary tumors and bone metastasis in women with HER-positive breast cancer.

Supplemental Figures

A total of 478 students from 8 disciplines, including social work, participated in an interprofessional education (IPE) Screening, Brief Intervention, and Referral to Treatment (SBIRT) project. A m...

HepG2 cells were incubated with a 16.5:1.7:1 ratio of cholesterol:sitosterol:campesterol (CSC), a ratio of the major sterols observed in the plasma of phytosterolemia patients, or with cholesterol alone in combination with [14 C]acetate for 24 h and the radioactivity incorporated into lipids determined. Cells incubated with CSC exhibited a 40% reduction in cholesterol esterification (p < 0.05) compared to cells incubated with cholesterol alone. In addition, a 17.5-fold reduction (p < 0.05) in total cholesterol (cholesterol plus cholesteryl ester) synthesis from [14 C]acetate was observed in cells incubated with CSC compared to cholesterol alone. Low-density lipoprotein receptor (LDLR) mRNA abundance was lower in cells incubated with CSC compared to cells incubated with cholesterol alone. Our results suggest that incubation of HepG2 cells with a ratio of sterols that mimic the plasma concentration seen in phytosterolemia patients reduces cholesterol esterification, total cholesterol synthesis, and inhibits LDLR mRNA abundance. We suggest that future cell and animal-based work on phytostosterolemia might employ this methodology to serve as a novel paradigm of the disease.

Introduction: PARP inhibitors have shown promising results in early studies for treatment of breast cancer susceptibility gene (BRCA)–deficient breast cancers; however, resistance ultimately develops. Furthermore, the benefit of PARP inhibitors (PARPi) in triple-negative breast cancers (TNBC) remains unknown. Recent evidence indicates that in TNBCs, cells that display “cancer stem cell” properties are resistant to conventional treatments, mediate tumor metastasis, and contribute to recurrence. The sensitivity of breast cancer stem cells (CSC) to PARPi is unknown. Experimental Design: We determined the sensitivity of breast CSCs to PARP inhibition in BRCA1-mutant and -wild-type TNBC cell lines and tumor xenografts. We also investigated the role of RAD51 in mediating CSC resistance to PARPi in these in vitro and in vivo models. Results: We demonstrated that the CSCs in BRCA1-mutant TNBCs were resistant to PARP inhibition, and that these cells had both elevated RAD51 protein levels and activity. Downregulation of RAD51 by shRNA sensitized CSCs to PARP inhibition and reduced tumor growth. BRCA1–wild-type cells were relatively resistant to PARP inhibition alone, but reduction of RAD51 sensitized both CSC and bulk cells in these tumors to PARPi treatment. Conclusions: Our data suggest that in both BRCA1-mutant and BRCA1–wild-type TNBCs, CSCs are relatively resistant to PARP inhibition. This resistance is mediated by RAD51, suggesting that strategies aimed at targeting RAD51 may increase the therapeutic efficacy of PARPi. Clin Cancer Res; 23(2); 514–22. ©2016 AACR.

BACKGROUND Alternatives to room temperature storage of platelets (PLTs) may be beneficial to extend the limited shelf life and support transfusion logistics in rural and military areas. The aim of this study was to assess the morphologic, metabolic, and functional aspects of PLTs stored at room temperature or in refrigerated conditions or cryopreserved. STUDY DESIGN AND METHODS A three-arm pool-and-split study was carried out using buffy coat–derived PLTs stored in 30% plasma/70% SSP+. The three matched treatment arms were room temperature stored (20-24°C), cold-stored (2-6°C), and cryopreserved (−80°C with dimethyl sulfoxide). Liquid-stored PLTs were tested over a 21-day period, while cryopreserved PLTs were examined immediately after thawing and after 6 and 24 hours of storage at room temperature. RESULTS Cold-stored and cryopreserved PLTs underwent a significant shape change, although the cryopreserved PLTs appeared to recover from this during subsequent storage. Glycolytic metabolism was reduced in cold-stored PLTs, but accelerated in cryopreserved PLTs, while oxidative phosphorylation was negatively affected by both storage conditions. PLT aggregation was potentiated by cold storage and diminished by cryopreservation in comparison to room temperature–stored PLTs. Cold storage and cryopreservation resulted in faster clot formation (R-time; thromboelastography), which was associated with an increase in microparticles. CONCLUSION Cold storage and cryopreservation of PLTs led to morphologic and metabolic changes. However, storage under these conditions appears to maintain or even enhance certain aspects of in vitro PLT function.

The goal of this dissertation was to study the impact of a co-learner on individuals’ motor performance, learning, and perceptions of the practice experience. In Experiment 1, we introduced “concurrent” practice, where partners practiced and observed one another simultaneously. Concurrent practice promoted movement coupling within pairs and was perceived as more interfering than individual and turn-taking practice of a balance-related task. However, these differences did not impact error during practice or testing. In Experiment 2, we studied whether matching or mismatching a partner is better for multi-skill learning. Partners practiced the same or different golf-putting skills in alternation. Although contextual interference would be higher for the mismatched group, mismatching did not modulate performance in practice or retention compared to matching or pure physical practice. In Experiments 3–5, we studied multi-skill learning when learners have control over how to practice. Experiment 3 tested self- versus peer-directed practice, when one partner practiced and the other passively observed or made task-switching decisions for the performer. Both self- and peer-schedulers made performance-dependent decisions, choosing to switch tasks based on timing error. Although peer-schedulers chose to switch more frequently, the groups did not differ in retention. In Experiments 4 and 5, we assessed the impact of a partner on self-directed practice choices, when partners switched turns after 9-trial blocks or after every trial. Self-directed learners showed partner-dependent practice, with the partner’s practice impacting sequence selection and switching frequency. Importantly, self-directed learners did not sacrifice the performance-dependent nature of their task-switching, suggesting that some practice behaviours are resistant to a partner’s practice while others are susceptible to modulation. Overall, this dissertation provides evidence that practice in pairs influences the practice decisions/behaviours of learners and provides efficiency benefits, as two people can be trained in the time otherwise devoted to one learner. However, practice in pairs did not improve learning compared to practice alone. Merely practicing with a partner, even if they exert some influence on decisions, was not enough to yield motor learning benefits for the individual. There is a need for well-powered studies to explore the conditions which might promote such benefits.

Almost all patients with EGFR-driven lung cancer who are treated with EGFR tyrosine kinase inhibitors (TKI) develop resistance to treatment. A single base (c.2369C>T) transition mutation, EGFR T790M, is the most frequent resistance event after first-generation exposure to EGFR TKIs. Whether T790M mutation is acquired or is selected from a preexisting clone has been a matter of significant debate. In this study, we show that treatment with EGFR TKIs leads to activation of the NFκB pathway, which in turn induces expression of activation-induced cytidine deaminase (AICDA). In turn, AICDA causes deamination of 5-methylcytosine to thymine at position c.2369 to generate the T790M mutation. Pharmacologic inhibition of the NFκB pathway or knockout of AICDA decreased the frequency or prevented the development of T790M mutation, respectively. In addition, patients treated with first-line EGFR TKI displayed increased expression of AICDA and detection of the T790M mutation upon progression. These results identify the mechanism of T790M acquisition and present an opportunity to target the process to delay or prevent it. Significance: These findings identify the mechanism behind acquisition of a common resistance mutation to TKI treatment in lung cancer.

Somatic mutations or deletions of TP53 and PTEN in ductal carcinoma in situ lesions have been implicated in progression to invasive ductal carcinomas. A recent molecular and mutational analysis of breast cancers revealed that inactivation of tumor suppressors, p53 and PTEN, are strongly associated with triple negative breast cancer. In addition, these tumor suppressors have important roles in regulating self-renewal in normal and malignant stem cells. To investigate their role in breast carcinogenesis, we knocked down these genes in human mammary cells and in non-transformed MCF10A cells. p53 and PTEN knockdown synergized to activate pro-inflammatory interleukin-6 (IL6)/Stat3/nuclear factor κB signaling. This resulted in generation of highly metastatic epithelial-to-mesenchymal transition-like cancer stem cells resulting in tumors whose gene expression profile mimicked that found in basal/claudin-low molecular subtype within the triple negative breast tumors. Constitutive activation of this loop in transformed cells was dependent on proteolytic degradation of suppressor of cytokine signaling 3 (SOCS3) resulting in low levels of this protein in basal/claudin-low cell lines and primary tumors. In non-transformed cells, transient activation of the IL6 inflammatory loop induced SOCS3 expression leading to pathway inactivation. In transformed cells, enforced expression of SOCS3 or interfering with IL6 pathway via IL6R blockade inhibited tumor growth and metastasis in mouse xenograft models. Furthermore, circulating tumor cells were significantly reduced in tumor-bearing animals when treated with anti-IL6R antibodies. These studies uncover important connections between inflammation and carcinogenesis and suggest that blocking pro-inflammatory cytokines may be utilized as an attractive strategy to target triple negative breast tumors, which currently lacks molecularly targeted therapies.

Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinomas. EGFR tyrosine kinase inhibitors (TKIs) are the mainstay of treatment for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, acquired EGFR mutations are the main mechanism of on-target resistance to TKIs. T790M mutation that occurs after first generation TKI treatment, is a cytosine to thymine (C>T) single nucleotide transition leading to a threonine to methionine amino acid change at position 790 (i.e. T790M). Our data suggest that resistant mutations are acquired events secondary to cytosine deamination through Activation Induced Cytosine Deamination enzyme (AICDA). Interestingly, treatment with Osimertinib, that overcomes the T790M mutation, leads to other acquired resistant mutations (C797S, G796S/R and L792F/H) that are mostly cytosine based. Results: Sub clones of the lung adenocarcinoma cell line PC9 with no evidence of T790M mutation by digital droplet PCR (ddPCR) at baseline, were treated with EGFR TKI. After serially increasing the treatment dose, T790M mutation was detected by ddPCR associated with a significant increase in AICDA expression. Similarly, when the resistant T790M PC9 clones were treated with Osimertinib, the expression of AICDA was also induced. Knocking down AICDA by shRNA or CRISPR-Cas9, decreases the development of T790M in PC9 cell lines after TKI exposure. Using mass spectrometry, we established that cytosine at codon 790 is methylated; thus, deamination of 5-methylcytosine leads to thymine directly, explaining the T790M C>T mutation. In addition, using ChIP assay and pharmacological inhibition we confirm that upon TKI exposure, NFκB binds AICDA promoter and induces its expression. In a mouse xenograft model, the induction of NFκB and AICDA after EGFR TKI exposure is abrogated by concurrent use of an NFκB inhibitor. Finally, patients treated with EFR TKI had an increased expression of AICDA upon progression. Conclusion: In EGFR driven lung adenocarcinoma, NFκB pathway is activated upon exposure to EGFR TKIs which induces AICDA expression. AICDA deaminates cytosine into other nucleotides leading to treatment resistance. Citation Format: Najwa El Kadi, Luo Wang, April Davis, Alexander Cooke, Varun Vadnala, Hasan Korkaya, Gregory P. Kalemkerian, Khaled A. Hassan. The T790M mutation is acquired through 5-methylcytosine deamination after EGFR TKI treatment in lung cancer [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 931.

Although current breast cancer treatment guidelines limit the use of HER2-blocking agents to tumors with HER2 gene amplification, recent retrospective analyses suggest that a wider group of patients may benefit from this therapy. Using breast cancer cell lines, mouse xenograft models and matched human primary and metastatic tissues, we show that HER2 is selectively expressed in and regulates self-renewal of the cancer stem cell (CSC) population in estrogen receptor-positive (ER+), HER2− luminal breast cancers. Although trastuzumab had no effects on the growth of established luminal breast cancer mouse xenografts, administration after tumor inoculation blocked subsequent tumor growth. HER2 expression is increased in luminal tumors grown in mouse bone xenografts, as well as in bone metastases from patients with breast cancer as compared with matched primary tumors. Furthermore, this increase in HER2 protein expression was not due to gene amplification but rather was mediated by receptor activator of NF-κB (RANK)-ligand in the bone microenvironment. These studies suggest that the clinical efficacy of adjuvant trastuzumab may relate to the ability of this agent to target the CSC population in a process that does not require HER2 gene amplification. Furthermore, these studies support a CSC model in which maximal clinical benefit is achieved when CSC targeting agents are administered in the adjuvant setting. Cancer Res; 73(5); 1635–46. ©2012 AACR.

R ecent case studies of lateral well completions have highlighted the apparent ineffectiveness of geometric stage performance with regards to fracture initiation and stimulated reservoir volume (Far et al., 2015; Ashton et al., 2013; Ganguly and Cipolla, 2012). It has been observed that poor stage performance can be attributed to varying rock type compositions along the borehole, due to lateral facies changes and/or wellbore porpoising. The inefficiencies linked to non or poor performing stages can result in higher costs, associated with equipment and materials, and limit the production potential of wells. This case study highlights how the application of an optimized hydraulic fracture stimulation plan, by honouring lateral well geological heterogeneities, improved completion efficiency and well production within the Cleveland Sandstone formation, Oklahoma, USA. Optimized completions Designing optimized hydraulic fracture stimulation in a lateral well requires an understanding of the near-well bore geomechanical properties and the near-well bore and farfield stresses along the entire lateral. Such reservoir characterization is normally developed from geomechanical and petrophysical analyses using wireline or logging-while-drilling (LWD) services that include acoustic and borehole- image logs. Unfortunately, economic considerations can often inhibit or prohibit the use of logging techniques and hence the complete characterization of the reservoir, especially in the current low oil and gas price environment. A practical and convenient alternative for reservoir characterization is to use the commonly available cuttings samples to provide gross characterization of a vertical or lateral well. Utilizing Automated Mineralogy (AM) techniques, such as CGG’s RoqSCANTM, (Ashton et al., 2013) provides comprehensive data from these cuttings which includes mineralogy, texture and rock properties throughout the entire length of the well. RoqSCAN analysis can be performed either in the laboratory or at the well site to provide near-real-time reservoir navigation and data to optimize the design of completions that will be performed soon after the well reaches total depth (TD).

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL-6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL-6 than parental cells. An IL-6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL-6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance.

The study of a zoonotic disease requires an understanding of the disease incidence in animal reservoirs. Rabies incidence in bats submitted to diagnostic laboratories does not accurately reflect the true incidence in wild bat populations as a bias exists for testing bats that have been in contact with humans or pets. This article details the rabies incidence in two species of bats collected from natural settings without such bias. In this study, brain smears from 0.6% and 2.5% of wild-caught and apparently healthy Tadarida brasiliensis and Eptesicus fuscus, respectively, were positive for rabies virus (RV) antigen. Conversely, 92% of the grounded T. brasiliensis were positive for RV. Serology performed on captive colony and sick bats reveal an immune response to rabies. This work illustrates the complex interplay between immunity, disease state, and the conundrum of RV maintenance in bats.

Summary Although breast cancer stem cells (BCSCs) display plasticity transitioning between quiescent mesenchymal-like (M) and proliferative epithelial-like (E) states, how this plasticity is regulated by metabolic or oxidative stress remains poorly understood. Here, we show that M- and E-BCSCs rely on distinct metabolic pathways and display markedly different sensitivities to inhibitors of glycolysis and redox metabolism. Metabolic or oxidative stress generated by 2DG, H2O2, or hypoxia promotes the transition of ROSlo M-BCSCs to a ROShi E-state. This transition is reversed by N-acetylcysteine and mediated by activation of the AMPK-HIF1α axis. Moreover, E-BCSCs exhibit robust NRF2-mediated antioxidant responses, rendering them vulnerable to ROS-induced differentiation and cytotoxicity following suppression of NRF2 or downstream thioredoxin (TXN) and glutathione (GSH) antioxidant pathways. Co-inhibition of glycolysis and TXN and GSH pathways suppresses tumor growth, tumor-initiating potential, and metastasis by eliminating both M- and E-BCSCs. Exploiting metabolic vulnerabilities of distinct BCSC states provides a novel therapeutic approach targeting this critical tumor cell population.

Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinomas. This leads to constitutive activation of EGFR, which triggers multiple downstream survival and proliferation pathways. EGFR tyrosine kinase inhibitors (TKIs) are the mainstay of treatment for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. Acquired EGFR mutations are the main mechanism of on-target resistance to TKIs. T790M mutation that occurs after first line TKI treatment, is a cytosine to thymine (C>T) single nucleotide transition leading to a threonine to methionine amino acid change at position 790 (i.e. T790M). Interestingly, treatment with Osimertinib, that overcomes the T790M mutation, leads to other acquired resistant mutations, C797S, G796S/R and L792F/H. Our data suggest that resistant mutations are acquired events secondary to cytosine deamination through Activation Induced Cytosine Deamination enzyme (AICDA). Results: Sub clones of the lung adenocarcinoma cell line PC9 with no evidence of T790M mutation by droplet digital PCR (ddPCR) at baseline, were treated with EGFR TKI. After serially increasing the treatment dose, T790M mutation was detected by ddPCR associated with a significant increase in AICDA expression. Knocking down AICDA by shRNA, decreases the development of T790M in PC9 cell lines after TKI exposure. Similarly, when the resistant T790M PC9 clones were treated with Osimertinib, the expression of AICDA was also induced. Using mass spectrometry, we established that cytosine at codon 790 is methylated; thus, deamination of 5-methylcytosine leads to thymine directly, explaining the T790M C>T mutation. In addition, using ChIP assay and pharmacological inhibition we confirm that upon TKI exposure, NFĸB binds AICDA promoter and induces its expression. In a mouse xenograft model, the induction of NFĸB and AICDA after EGFR TKI exposure is abrogated by concurrent use of an NFĸB inhibitor. Finally, patients treated with EFR TKI had an increased expression of AICDA upon progression. Conclusion: In EGFR driven lung adenocarcinoma, NFĸB pathway is activated upon exposure to EGFR TKIs which induces AICDA expression. AICDA deaminates cytosine into other nucleotides leading to treatment resistance. Citation Format: Najwa El Kadi, April Davis, Alexander Cooke, Luo Wang, Hasan Korkaya, Gregory Kalemkerian, Khaled Hassan. Activation Induced Cytosine Deamination, AICDA, is induced after EGFR TKI exposure leading to secondary resistant mutations in lung adenocarcinoma [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 5836.

The alphaherpesviral envelope protein pUS9 has been shown to play a role in the anterograde axonal transport of herpes simplex virus 1 (HSV-1), yet the molecular mechanism is unknown. To address this, we used an in vitro pulldown assay to define a series of five arginine residues within the conserved pUS9 basic domain that were essential for binding the molecular motor kinesin-1. The mutation of these pUS9 arginine residues to asparagine blocked the binding of both recombinant and native kinesin-1. We next generated HSV-1 with the same pUS9 arginine residues mutated to asparagine (HSV-1pUS9KBDM) and then restored them being to arginine (HSV-1pUS9KBDR). The two mutated viruses were analyzed initially in a zosteriform model of recurrent cutaneous infection. The primary skin lesion scores were identical in severity and kinetics, and there were no differences in viral load at dorsal root ganglionic (DRG) neurons at day 4 postinfection (p.i.) for both viruses. In contrast, HSV-1pUS9KBDM showed a partial reduction in secondary skin lesions at day 8 p.i. compared to the level for HSV-1pUS9KBDR. The use of rat DRG neuronal cultures in a microfluidic chamber system showed both a reduction in anterograde axonal transport and spread from axons to nonneuronal cells for HSV-1pUS9KBDM. Therefore, the basic domain of pUS9 contributes to anterograde axonal transport and spread of HSV-1 from neurons to the skin through recruitment of kinesin-1. IMPORTANCE Herpes simplex virus 1 and 2 cause genital herpes, blindness, encephalitis, and occasionally neonatal deaths. There is also increasing evidence that sexually transmitted genital herpes increases HIV acquisition, and the reactivation of HSV increases HIV replication and transmission. New antiviral strategies are required to control resistant viruses and to block HSV spread, thereby reducing HIV acquisition and transmission. These aims will be facilitated through understanding how HSV is transported down nerves and into skin. In this study, we have defined how a key viral protein plays a role in both axonal transport and spread of the virus from nerve cells to the skin.

Developmental pathways such as Notch play a pivotal role in tissue-specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch+) or reduced activity (Notch−) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays, we investigated the role of the Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expression of breast CSC markers. Interestingly Notch+ cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notch+ cells demonstrated tumor initiation capacity at serial dilutions in mouse xenografts, whereas Notch− cells failed to generate tumors. γ-Secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent, effectively targets these Notch+ cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our study revealed a molecular mechanism for the role of Notch-mediated regulation of breast CSCs and provided a compelling rationale for CSC-targeted therapeutics. Mol Cancer Ther; 14(3); 779–87. ©2015 AACR.

EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism. Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 15% of lung adenocarcinomas. This leads to constitutive activation of EGFR, which triggers multiple downstream survival and proliferation pathways. Currently, EGFR tyrosine kinase inhibitors (TKIs) are first line therapy for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. Despite initial significant response to TKIs, most tumors develop resistance. The main mechanism of resistance detected in 50-60% of cases is a cytosine to thymine (C>T) single nucleotide transition mutation at position 2369. This causes a threonine to methionine amino acid change at position 790 (i.e. T790M). Our data suggests that the C>T mutation is an acquired event secondary to cytosine deamination by Activation Induced Cytosine Deamination enzyme (AICDA). Results: Single cell clones of lung adenocarcinoma cell line, PC9, were treated with EGFR TKI. At baseline, these clones have EGFR exon 19 deletion but no evidence of T790M mutation by digital droplet PCR (ddPCR). However, after treatment with a serial increasing dose of EGFR TKI, T790M mutation was detected by ddPCR. Assessing whether cytosine deamination enzymes were altered by this treatment, a significant increase in AICDA expression was seen. Furthermore, recombinant AICDA protein could deaminate cytosine at position 2369 in vitro. In addition, using mass spectrometry and methylation specific primers, we determined that cytosine at position 2369 is in fact methylated. This further supports our hypothesis since 5-methyl cytosine is deaminated into thymine directly. Since in germinal center B-lymphocytes, AICDA is activated through a non-canonical NFkB mechanism, we assessed NFkB pathway in PC9 cell line. RelB and p52 expression were significantly increased after TKI treatment. In addition direct interaction between RelB and AICDA promoter was confirmed by ChIP Assay. These findings were also seen in a mouse PC9 xenograft model. Daily oral gavage of EGFR TKI caused significant increase in the expression of RelB as well as AICDA. Adding NFkB inhibitor twice weekly inhibited the expression of RelB and AICDA. Finally, knocking down AICDA by shRNA prevented the development of T790M mutation in PC9 cell lines after TKI exposure. Conclusion: Our data suggest that the T790M mutation could be actively acquired after TKI treatment through a cytosine deamination process by AICDA. This would have significant implications for treatment with targeted therapy. In fact, Imatinib resistance in CML and GIST tumors have a similar C>T single nucleotide transition mutation. Citation Format: Khaled Hassan, Najwa El Kadi, April Davis, Gregory Kalemkerian, Luo Wang, Hasan Korkaya. The development of EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism [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 4119. doi:10.1158/1538-7445.AM2017-4119

Background: Triple negative breast cancers (TNBCs) have a poor prognosis and are a therapeutic challenge due to resistance to multiple chemotherapy drugs. Growing evidence suggests that cancer stem cells (CSCs) may be promising therapeutic targets for treating TNBCs. Poly ADP ribose polymerase (PARP) inhibitors have shown striking activity in preclinical models of BRCA-deficient breast carcinomas. However they are less efficacious in tumors without germline BRCA mutations, which account for more than 80% of all TNBCs. Our data demonstrates that treatment of TNBC with PARP inhibitor increases CSCs, likely due to an elevated homologous recombination (HR) repair mechanism. Several small studies have suggested that histone deacetylase (HDAC) inhibitors, which decrease HR repair proteins, may sensitize non-BRCA-mutated TNBC to PARP inhibitors. Currently, little is known whether this synergistic effect can reverse the increase in CSCs caused by PARP inhibition. In this study, our aim was to investigate if HDAC inhibitors could sensitize CSCs to PARP inhibitors in TNBCs. Results: Using four TNBC cell lines, we confirmed the synthetic lethality of HDAC and PARP inhibitors (Vorinostat and Olaparib respectively) in vitro. CompuSyn analysis revealed Combination Indices of 0.66, 0.44, 0.47 and 0.16 in SUM159, MDAMB231, SUM149 and HCC1937 cells respectively at ED50. An ALDEFLUOR assay revealed ∼1.9 fold increase in the CSC population after PARP inhibition in BRCA-mutant SUM149 and HCC1937 cells, while the absolute CSC number, (total cell number times percentage of ALDEFLUOR-positive cells) remained unchanged. Whereas in BRCA-wild type TNBC cell lines SUM159 and MDAMB231, there was no change in CSCs. Addition of the HDAC inhibitor decreased absolute CSC number by 85%, 78% and 40% in SUM149, SUM159 and HCC1937 cells respectively after 7 days of PARP inhibition. Although the mechanism is not fully understood at present, we demonstrated that Rad51, a key player in HR repair, mediates the sensitivity of TNBCs to PARP inhibition, and HDAC inhibitor prevents the formation of Rad51 foci at DNA damage site. This may indicate that HDAC inhibition sensitizes the CSCs of TNBCs to PARP inhibition via suppressing the HR pathway. Conclusion: Our data suggests that PARP inhibition only targets the bulk cancer cells in BRCA-mutant TNBC cells while sparing CSCs and has no effect on BRCA WT cells. HDAC inhibition sensitized CSCs of TNBC to PARP inhibition regardless of BRCA status, possibly via interfering with Rad51 function. The inhibitors we used in the present study are either FDA-approved or at Phase III clinical trials, and the combination is predicted to have low toxicity. Thus, our findings are promising and could be rapidly translated into clinical use. Citation Format: Yajing Liu, Rachel Martin-Trevino, Li Shang, April Davis, Max Wicha, Suling Liu, Monika Burness. Histone deacetylase inhibitors sensitize cancer stem cells to PARP inhibitors in triple-negative breast cancer. [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 4226. doi:10.1158/1538-7445.AM2015-4226

Cancer stem cells (CSCs) drive tumor growth as well as mediating metastasis and treatment resistance. In breast cancer (BC), CSCs exist in alternative mesenchymal (EMT) and epithelial (MET)-like states characterized by the expression of CD44+CD24- and aldehyde dehydrogenase (ALDH) respectively. BCSCs display phenotypic plasticity allowing them to transition between EMT and MET states in a process regulated by the tumor microenvironment. The plasticity of BCSCs suggests that it may be necessary to simultaneously target alternative BCSC states to achieve maximal eradication of these cell populations. In order to develop strategies to target these BCSC states, we measured the cellular bioenergetics of EMT and MET BCSCs compared to non-stem bulk tumor cells as well as the sensitivity of distinct BCSC states to inhibitors of glucose and hydroperoxide metabolism. Using a Seahorse XCF instrument, we found that both MET and EMT BCSCs display higher glycolytic potential than bulk tumor cells. Increased glycolysis correlated with elevated expression of hexokinase 2, a rate limiting glycolytic enzyme in cancer. Interestingly, the glycolytic inhibitor, 2-deoxyglucose (2DG), specifically inhibited EMT BCSCs in a dose dependent fashion; however, MET BCSCs were completely refractory to this treatment. Proteomic and RNA-seq analyses revealed that two important arms of hydroperoxide metabolism, the thioredoxin- and glutathione-mediated antioxidant pathways, were robustly up-regulated in MET BCSCs, suggesting that these cells are rendered resistant to glycolysis inhibition via an enhanced anti-oxidant defense. Pharmacologic inhibition of this antioxidant defense by Auranofin (an inhibitor of thioredoxin reductase) is sufficient to deplete MET BCSCs in SUM149 BC cells which were rescued by NAC, a ROS scavenger, or catalase, a specific enzyme capable of degrading intracellular H2O2. Finally, administration of 2DG together with Auranofin and BSO (an inhibitor of glutathione metabolism) synergistically suppressed tumor growth in a patient derived xenograft (PDX) model by suppressing both EMT and MET BCSCs. This study suggests that utilizing metabolic inhibition to simultaneously target EMT and MET BCSCs is a viable therapeutic strategy with important clinical implications. Citation Format: Ming Luo, April Davis, Sean McDermott, Evelyn Jiagge, Michael Brooks, Elizabeth Gheordunescu, Tahra Luther, Shawn G. Clouthier, Sarah Conley, Douglas R. Spitz, Max S. Wicha. Targeting EMT and MET breast cancer stem cell states through simultaneous inhibition of glycolytic and antioxidant pathways. [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 2311. doi:10.1158/1538-7445.AM2015-2311

Big brown (Eptesicus fuscus) and Mexican free-tailed (Tadarida brasiliensis) bats were inoculated with the New York 99 strain of West Nile virus to assess their potential to serve as amplifying hosts and determine the clinical effect of infection. Groups of three or four bats were bled at daily intervals between 1 and 6 days after inoculation to determine the pattern of viremia. Beginning 2 days after inoculation, virus was isolated each day from one or more E. fuscus bats, in titers ranging from 10 to 180 plaque-forming units per milliliter of serum. Virus was not isolated from any of the sera collected from T. brasiliensis bats. None of the bats from either species showed clinical signs associated with exposure to virus. Sera from an additional 149 bats collected in Louisiana in 2002 during an epizootic of West Nile fever were tested for antibodies to virus, and two were found to be positive. These data suggest that bats from these two widely distributed species are unlikely to serve as amplifying hosts for West Nile virus.

We demonstrated that knock-down of tumor suppressors p53 and PTEN in mammary epithelial MCF10A cells (called MCF10A-p53-PTEN-) results in generation of aggressive tumors. Furthermore MCF10A-p53-PTEN- cells also display an increased EMT phenotype as well as expansion of cancer stem cell (CSC) population. Utilizing Affymetrix gene expression analyses, we confirmed that malignant MCF10Ap53-PTEN- cells displayed a gene expression pattern which closely resembled that of the basal/claudin molecular subtype with the TN. This subtype is previously characterized by the expression of EMT and stem cell genes. The Inflammatory cytokines IL6 and TGF-β have been found to regulate stem cells and EMT. Consistent with these data, we found that MCF10A-p53-PTEN- cells secreted significantly higher levels of IL6 (>1000 fold) and TGF-β (>200 fold) compared to parental or single gene deleted cells. Furthermore combined deletion of p53 and PTEN compared to single gene knockdown resulted in transformation of MCF10A cells generating aggressive metastatic tumors in NOD/SCID mice. IL6 is a key regulator of inflammatory responses and orchestrates these physiological functions by controlling the SOCS3 mediated Stat3/NF-κB pathway. Consistent with these reports, we demonstrated that SOCS3 is abundantly expressed in MCF10A cells where it negatively regulates the IL6 feedback loop. Unexpectedly, we found that SOCS3 protein was undetectable in MCF10A-p53-PTEN- cells. Basal/claudin-low breast cancer cell lines that display an activated IL6 feedback loop also had low SOCS3 protein levels. Furthermore, we found a similar discordance between SOCS3 protein and mRNA levels in primary basal/claudin-low breast cancer samples as demonstrated by immunochemistry utilizing a primary breast cancer tissue array. Together these findings suggest that loss of SOCS3 protein expression allows for maintenance of the IL6 mediated inflammatory feedback loop in aggressive/metastatic TN breast cancers. We found that MCF10A-p53-PTEN- cells displayed increased proteasome activity as evidenced by SOCS3 protein accumulation upon proteasome inhibition. Proteolytic degradation of SOCS3 protein in turn is controlled by constitutive activation of the IL6 mediated Stat3/NF-κB pathway. We demonstrate that enforced expression of SOCS3 or IL6 pathway inhibition interfering with this loop through IL6R blockade repress the CSC population, reducing the tumor growth and metastasis in mouse xenografts. These studies provide a strong rationale for development of IL6 pathway-targeting agents for the treatment of TNBC, an aggressive disease that currently lacks molecularly targeted therapeutics. Citation Format: Hasan Korkaya, Maria Ouzounova, Gwangil Kim, Ali Quraishi, April Davis, Nader Tawakkol, Sumeyye Korkaya, Max S. Wicha. SOCS3-mediated regulation of inflammatory cytokines in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3869. doi:10.1158/1538-7445.AM2014-3869

Recent studies by us and others suggested that activation of inflammatory feedback loop play critical role in metastasis and therapeutic resistance by regulating EMT/CSC phenotype. Of these cytokines, IL6 is a key regulator of inflammatory responses and orchestrates these physiological functions by controlling the Stat3/NF-kB pathway which is negatively regulated by the suppressor of cytokine signaling 3 (SOCS3). Consistent with these reports, we demonstrated in our preliminary data that SOCS3 is abundantly expressed in non-malignant MCF10A cells as well as in luminal and HER2+ tumors where it negatively regulates the IL6/Stat3/NF-kB signaling. In contrast, SOCS3 protein was undetectable in basal cluadin-low breast cancer cell lines and primary tumors which express substantially higher levels of IL6. We developed transformed MCF10A model by simultaneous knockdown of p53 and PTEN. In this model single p53 and PTEN deletions result in hyperplasia or DCIS-like lesions. Interestingly, SOCS3 protein expression was undetectable in MCF10A-p53-PTEN- cells compared to parental MCF10A, single p53 (MCF10A-p53-) or PTEN (MCF10A-PTEN-) deleted cells. We demonstrated that IL6 mediated Stat3/NF-kB signaling is negatively regulated by SOCS3 that strongly inhibited the IL6/Stat3/NF-kB pathway when overexpressed in MCF10A-p53-PTEN- cells. Moreover, enforced SOCS3 expression in these cells reduced the tumor growth and inhibited metastasis. Furthermore, loss of SOCS3 expression is strongly associated with increased risk of recurrent disease in breast cancer patients. We hypothesized that loss of SOCS3 in TNBCs results in activation of IL6 feedback loop which drives aggressive metastatic phenotype. Interestingly Stat3/NF-kB signaling drives expression of SOCS3 in response to inflammation and serves as negative feedback loop, although molecular mechanism of SOCS3 expression in cancer has not been investigated. IL6 signaling has been previously reported to be involved in epigenetic regulation. We therefore correlated the gene expression and methylation profile after enforced SOCS3 expression. Our analysis revealed that expression levels of a significant number of genes involved in transcription, migration and metabolism were reverted to those of the parental non transformed cells and their transcription was regulated by methylation. These results suggest that interplay between genetic and epigenetic changes during malignant transformation of mammary epithelial cells may activate multiple signaling cascades and reconstitution of SOCS3 may have therapeutic utility in TNBC. Citation Format: Maria Ouzounova, Gwangil Kim, April Davis, Ahmed A. Quraishi, Nader Tawakkol, Shalini Kota, Max S. Wicha, Hasan Korkaya. Inactivation of p53/PTEN confers a specific epigenetic profile regulated by IL6-SOCS3 signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3866. doi:10.1158/1538-7445.AM2014-3866

Background: Despite recent advances in treatment for some subtypes of breast cancer (BC), triple negative (TN) BC remains a therapeutic challenge due to a lack of effective targeted agents. TNBC accounts for 10-17% of all breast malignancies and is associated with younger age, more advanced stage at diagnosis, and poorer outcome when compared with other BC subtypes. Growing evidence supports that human breast cancers are driven by a small subpopulation of cells termed cancer stem cells (CSCs) with greater tumorigenic potential. CSCs contribute to tumor initiation, maintenance, metastasis, therapeutic resistance and recurrence; thus targeting CSCs is a promising therapeutic intervention. Our laboratory has previously demonstrated the importance of the inflammatory signaling molecules interleukin 6 (IL-6) and 8 (IL-8) in maintaining CSCs in several BC subtypes. Despite promising results in other subtypes, targeting IL-6 or IL-8 alone has had less success in TNBC. Thus, this study explores the effect of combination therapy consisting of dual inhibition of IL-6 and IL-8 and chemotherapy on CSCs in TNBCs. Results: Our preliminary results demonstrate that docetaxel treatment induces three and five-fold increases in IL-6 and IL-8 levels, respectively, in an in vitro culture system of the TNBC cell line SUM159, most likely due to cell death. However, addition of IL-6 or IL-8 inhibitors does not change the cytokine levels or CSC population compared to docetaxel treatment alone. We hypothesized that the tumor microenvironment is critical to regulate the effect of cytokines on tumor growth; we therefore tested our treatment regimen in a xenograft model by injecting SUM159 cells into mammary fat pad of female NOD/SCID mouse. Docetaxel treatment alone slightly reduced tumor weight compared to vehicle control (0.6±0.4g v.s. 1.07±0.4g, p=0.04), as expected. No change was seen in tumors treated with docetaxel and IL-8 inhibitor (0.8±0.2g). Docetaxel in combination with either IL-6 inhibition (0.2±0.1 g) or IL-6 and IL-8 inhibitors (0.3±0.07 g) completely blocked tumor progression during four weeks of treatment (p=0.008, p=0.01 respectively compared to vehicle control). ALDEFLUOR assay was used to quantitate CSCs on these xenografts. The combination treatment of docetaxel plus IL-6 and IL-8 inhibitors decreased the CSC population compared to both vehicle control (0.7±0.3% v.s. 2.6%±0.8%, p=0.01) and docetaxel alone (0.7±0.3% v.s 2.6±0.3%, =0.001). Discussion: Our results suggest that IL-6 and IL-8 inhibition in combination with chemotherapy can inhibit growth of TNBC possibly by targeting the CSC population. The addition of IL-6 and IL-8 inhibition to chemotherapy may be a promising therapeutic approach for treating TNBC patients. Citation Format: Yajing Liu, Leonel Hernandez-Aya, April Davis, Max Wicha, Monika Burness. Interleukin 6 and interleukin 8 inhibitors, when used in combination with docetaxel, inhibit tumor progression in mouse model of triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3893. doi:10.1158/1538-7445.AM2014-3893

Although current breast treatment guidelines limit the use of HER2 blocking agents such as trastuzumab to women whose tumors display HER2 gene amplification, recent retrospective analysis by several groups have suggested that in the adjuvant setting a wider group of patients whose tumors do not display HER2 amplification may also benefit from this therapy. We hypothesized that these unanticipated results might be explained by the cancer stem cell (CSC) hypothesis. We previously demonstrated that HER2 was an important regulator of CSCs in HER2 amplified breast cancers. We now utilize breast cancer cell lines, mouse xenograft and matched human primary and metastatic tumor samples to demonstrate that HER2 is selectively expressed in and regulates the self-renewal of CSC populations in ER-positive luminal breast cancers which do not display HER2 amplification. In luminal MCF7 and ZR-75-1 breast cancer cells, as well as in primary human tumors, we demonstrate an association between cells which express HER2 and the stem cell marker aldehyde dehydrogenase (ALDH1). In vitro trastuzumab reduced the CSC population as assessed by tumorsphere formation or ALDH expression while no such effects were scene in basal/claudin-low MDA-MB-231 and SUM159 cells. In mouse luminal tumor xenografts, the effects of trastuzumab were highly dependent on the timing of administration. While the effects of trastuzumab on established tumors were limited to those with HER2 amplification, trastuzumab administration begun immediately after tumor inoculation blocked the growth of luminal tumors which did not display HER2 amplification. HER2 expression was increased in luminal tumors grown in mouse bone xenografts, as well as in bone metastasis from patients with luminal breast cancer compared to matched primary tumors. Furthermore, this increase in HER2 protein expression was not due to gene amplification, but rather was mediated by RANK ligand in the bone microenvironment. These studies suggest that the clinical efficacy of adjuvant trastuzumab may relate to the ability of this agent to target the CSC population in a process that does not require HER2 gene amplification. Furthermore, these studies support a CSC model in which maximal clinical benefit is achieved when CSC targeting agents are administered in the adjuvant setting. Citation Format: Korkaya Hasan, Fayaz Malik, Kathleen C. Day, Suthinee Ithimakin, April Davis, Ahmed A. Quraishi, Nader Tawakkol, Kathleen Woods Ignatoski, Dafydd Thomas, Shawn G. Clouthier, Max S. Wicha. Regulation of luminal breast cancer stem cells by HER2 does not require gene amplification providing a potential molecular explanation for the efficacy of adjuvant trastuzumab in patients with “clinically HER2-negative” breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4065. doi:10.1158/1538-7445.AM2013-4065

Backgroung: The Epithelial-Mesenchymal Transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. EMT cells possess the characteristics of cell motility, invasiveness, and have stem cells properties. EMT expression profile correlates with resistance to chemotherapy and poor outcome in multiple tumors and there is evidence suggesting an integral role of Notch pathway in mediating an EMT status. In this study, we investigated Notch activity and EMT status and the correlation with chemoressitance in lung adenocarcinoma. Results: Transduction of multiple lung adenocarcinoma cell lines, NCI-H1299, NCI-H460, NCI-H358, NCI-H441 with a Notch GFP-reporter construct identified a subset of cells with high Notch activity (GFP-positive). We compared cell migration capacity of GFP-positive and -negative cells utilizing a transwell invasion assay. Cells with high Notch activity, as evidenced by GFP expression, had a significantly higher migration capability as compared to GFP-negative cells. To evaluate resistance to chemotherapy, we treated GFP-pos and GFP-neg cells with docetaxel or cisplatin and measured apoptosis by Annexin V expression with flow cytometry. GFP-pos cells showed major resistance to both agents as compared to GFP-neg cells. When GFP-pos cells were exposed to a Notch activity blocker, gamma secretase inhibitor (GSI), these cells were rendered sensitive to cisplatin or docetaxel exposure as evident by an increase in Annexin V expression. Using RNA microarray analysis and RT-PCR assays, there was an increased expression of EMT related genes (Vimentin, N-Cadherin, Snail-1,….) in GFP-pos cells as compared to GFP-neg cells. Furthermore, NOD/SCID mice with subcutaneous primary lung tumor xenografts treated with GSI showed decreased expression of downstream effectors of Notch as as well as vimentin. Mice treated with combination of GSI and docetaxel had better tumor control as compared to those treated with GSI and docetaxel as single agents. Conclusion: These studies suggest that the Notch pathway is an important regulator of EMT status and that its inhibition induces mesenchymal to epithelial transition overcoming chemoresistnace. Notch inhibitors in combination with standard chemotherapy present a potential effective approach to achieve better disease control in lung cancer. Citation Format: Khaled Hassan, Luo Wang, Hasan Korkaya, Paige O'Dowd, Sarah Dubaisi, Rayyan Kadi, Rawad Nasr, Binghui Zhang, April Davis, Gregory Kalemkerian, Max Wicha. Blocking the Notch pathway induces mesenchymal-to-epithelial transition in lung adenocarcinoma: A novel approach to overcoming chemoresistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 960. doi:10.1158/1538-7445.AM2013-960

The development of HER2 targeting agents such as trastuzumab and lapatinib represents one of the greatest achievements in clinical oncology demonstrating the effectiveness of molecularly targeted therapeutics (2). Despite the clinical benefits of trastuzumab in improving progression free and overall survival, majority of patients demonstrate “intrinsic” resistance or develop “acquired” resistance within one-to-two years. Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance remain to be investigated. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrated that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of breast cancer stem cell (CSC) population. We and others previously demonstrated that cytokines including IL6 and IL8 regulates breast cancer stem cell self-renewal. Furthermore, elevated serum levels of these cytokines in breast cancer patients strongly correlated with metastatic disease and therapeutic resistance. We therefore hypothesized that activation of IL6 feedback loop may mediate trastuzumab resistance. Utilizing these models, we demonstrated that long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. Recombinant IL6 or conditioned medium from high-IL6 secreting cells resulted in trastuzumab resistance in parental PTEN wild type cells expanding CSCs with EMT phenotype characterized by the CD44+/CD24- expression and EMT gene signature. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies have important clinical implications demonstrating that trastuzumab resistance may be mediated by an IL-6 inflammatory loop and suggest that blocking this loop represents a novel strategy to overcome trastuzumab resistance. . Citation Format: April L. Davis, Hasan Korkaya, Maria Ouzounova, Fayaz Malik, Gwangil Kim, Ahmed A. Quraishi, Suling Liu, Shawn G. Clouthier, Max S. Wicha. IL6 feedback loop regulates breast cancer stem cells which mediate trastuzumab resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3736. doi:10.1158/1538-7445.AM2013-3736

Background: The Epithelial-Mesenchymal Transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. EMT cells have stem cells properties and possess the characteristics of cell motility, invasiveness and chemotherapy resistance. EMT expression profile correlates with poor outcome in multiple tumors and there is evidence suggesting an integral role of Notch pathway in mediating EMT. In this study, we investigated Notch activity and EMT status and the correlation with chemoressitance in non-small cell lung cancer (NSCLC). Results: Transduction of multiple NSCLC cell lines, NCI-H1299, NCI-H460, NCI-H358, NCI-H441 with a Notch GFP-reporter construct identified a subset of cells with high Notch activity (GFP-pos). We compared the tumor invasivness capacity of GFP-pos and GFP-neg cells utilizing a transwell invasion assay. Cells with high Notch activity, as evidenced by GFP expression, had multiple fold higher invasion capability compared to GFP-neg cells (p=0.0001). To evaluate whether Notch activity correlates with resistance to chemotherapy, we treated GFP-pos and GFP-neg cells with docetaxel or cisplatin and assessed Annexin V expression using flow cytometry. GFP-pos cells had five fold less apoptotic cells as compared to GFP-neg cells indicating resistance of GFP-pos cells to chemotherapy. When GFP-pos cells were exposed to a Notch signaling inhibitor inhibitor, gamma secretase inhibitor (GSI), these cells were rendered sensitive to cisplatin and docetaxel. RNA microarray analysis and RT-PCR analysis revealed an increased expression of EMT related genes (Vimentin, N-Cadherin, Snail-1,….) in GFP-pos cells as compared to GFP-neg cells. Furthermore, NOD/SCID mice with subcutaneous lung tumor xenografts treated with GSI showed decreased expression of downstream effectors of Notch as well as vimentin. This combination of GSI and docetaxel reduced tumor grwoth in mouse xenographs compared to GSI and docetaxel as single agents. Conclusion: These studies suggest that the Notch pathway is an important regulator of EMT status and that its inhibition may alter EMT chemo-resistance properties. The addition of a Notch inhibitor may enhance the efficacy of chemotherapy in the treatment of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3309. doi:1538-7445.AM2012-3309

The PTEN and p53 tumor suppressor genes, frequently inactivated in human breast cancers have been shown to be regulators of normal and malignant breast stem cell self-renewal. In addition, inactivation of tumor suppressors in preneoplastic lesions are implicated in tumor progression from ductal situ in carcinoma (DCIS) to invasive ductal carcinoma. To determine the relationship between stem cell self-renewal and carcinogenesis, we examined the effects of PTEN and p53 knockdown on normal mammary epithelial cells obtained from reduction mammoplasties, as well as in the non-transformed mammary cell line MCF10A. Simultaneous knockdown of PTEN and p53 in normal mammary epithelial cells generated hyperplastic and DCIS-like lesions when these cells were transplanted into the humanized mammary fat pads of NOD/SCID mice. In MCF10A cells knockdown of PTEN and p53 synergized to expand the stem cell compartment transforming these cells and generating highly metastatic tumors in NOD/SCID mice. Stem cell expansion was driven by activation of an inflammatory loop involving both IL-6 and TGF-b. This inflammatory loop was mediated by both AKT/b-Catenin and Stat3/NF-kB pathways. Conversely collective inhibition of these pathways including TGF-b, IL-6 and Wnt/b-Catenin and Stat3/NF-kB was necessary to revert back this stem cell phenotype. This may suggest that synergistic activity of these pathways may contribute to the expansion and transformation of mammary stem cells. The expanded cancer stem cells were highly metastatic and invasive and displayed properties of EMT. The IL6-R antibody tocilizumab blocked this loop resulting in a reduction of CSCs and thus inhibiting tumor growth in mouse xenografts. These studies suggest that loss of PTEN and p53 transforms mammary epithelial cells by activating an inflammatory loop mediated by IL-6 and TGF-b, generating CSCs with an EMT phenotype. Since CSCs drive tumor growth and metastasis, targeting the self-renewal by interfering with this CSC, inflammatory loop represents a rational strategy for breast cancer prevention and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3349. doi:1538-7445.AM2012-3349