• The Effect of Drug Pressure and Transmission Setting on Sulfadoxine-Pyrimethamine Resistant Plasmodium falciparum Haplotype Prevalence and Selective Sweep Characteristics, in Malawi

      Artimovich, Elena M.; Takala-Harrison, Shannon (2014)
      Background: The continued expansion of resistance to anti-malarial chemotherapies is a threat to public health, and to malaria control and elimination. The reexpansion of drug sensitive parasites after the removal of drug pressure has renewed interest in epidemiological factors affecting resistance haplotype dynamics, in the hopes that previously abandoned drugs might once again find clinical utility. Objectives: Estimate the effect of changes in drug pressure and different malaria transmission settings on sulfadoxine-pyrimethamine (SP)-resistant haplotype prevalence and characteristics of selective sweeps. Methods: DNA was extracted from dried blood spots representing malaria infections from three time periods (high-SP use 1999-2001, transition-period 2007-2008, low-SP use 2012) of drug pressure in Malawi and three transmission settings (urban-low, rural-moderate, rural-high). Pyrosequencing and microsatellite genotyping were performed on all samples to determine haplotype prevalence and sweep characteristics. Changes in haplotype prevalence were assessed via Chi-squared tests and changes in sweep characteristics via permutation. Results: We observed the persistence of the DHFR 51I/59R/108N and DHPS 437G/540E haplotypes, five years after reduction in SP pressure as well as an increase in the prevalence of DHPS 437G/540E/581G haplotype. Selective sweeps indicated little to no fitness cost to the DHFR 51I/59R/108N and DHPS 437G/540E haplotypes in the absence of strong SP pressure. A decline in polyclonal infections was found across the three time periods. No significant difference in haplotype prevalence was found between transmission settings. Sweep characteristics could suggest divergent evolutionary history in the rural-moderate transmission setting. Conclusions: There is little to no fitness cost of SP-resistance in the absence of strong SP pressure in these three transmission settings within Malawi. The reexpansion of SP sensitive parasites in the region is not expected under current epidemiological conditions. Reduction in the amount of malaria in the region could further reduce the likelihood of reexpansion through the elimination of rare haplotypes due to genetic drift.
    • Molecular marker of Plasmodium falciparum resistance to chloroquine: Implications for malaria control and anti-malarial immunity

      Djimde, Abdoulaye; Plowe, Christopher V. (2001)
      Malaria is a major public health problem in tropical countries and is becoming a threat to global health. Resistance of Plasmodium falciparum to chloroquine, the cheapest, safest and most widely used antimalarial drug is now widespread. The basic mechanisms involved are just beginning to be understood. Three genes, pfcrt, cg2 and pfmdr 1 encoding PfCRT, CG2 and Pgh1, respectively, have been associated with chloroquine resistance in laboratory lines of Plasmodium falciparum. The role of these genes in field chloroquine resistance is not known. We conducted prospective efficacy trials in Mali (West Africa) to collect rigorously characterized resistant and sensitive isolates of Plasmodium falciparum. PCR based molecular methods were developed to detect gene polymorphisms and assess their selection by chloroquine and association with treatment outcome. We show that resistance-conferring polymorphisms of all three genes were selected in vivo. However, chloroquine resistant parasites carrying wild-type polymorphisms were found for all genes but pfcrt. The presence of PfCRT K76T mutation was the most strongly associated with clinical chloroquine resistance (OR, 18.8; 95% CI 6.5-58.3). The data confirm PfCRT's principal role in chloroqine resistance and demonstrate that the K76T mutation is the best available molecular marker of chloroquine resistance. To assess PfCRT K76T as a tool for monitoring chloroquine resistance, we conducted similar surveys in three sites with different malaria epidemiological characteristics and calculated genotype-resistance indices (GRI) for each site. Although the prevalence of the PfCRT 76T was consistently higher than clinical chloroquine resistance, GRIs adjusted for age were between 1.6 and 2.7. This GRI model should permit the determination of rates of clinical chloroquine resistance in large regions based on simple molecular surveys of prevalence of PfCRT 76T, without the need for lengthy and expensive in vivo studies. Because most patients cleared PfCRT K76T mutant parasites we hypothesized that host factors accounted for this phenomenon. We show that the ability to clear these mutant parasites is strongly associated with age, which is the only accepted marker of protective immunity in malaria. We propose the clearance of PfCRT mutant parasites as a model system for the investigation of antimalarial immunity.
    • Overcoming Drug Resistance in BRaf Mutated Melanoma Cells

      Samadani, Ramin; Shapiro, Paul, Ph.D. (2015)
      Targeted inhibition of the ERK1/2 signaling pathway has received much attention after the transient success of the BRaf inhibitor, vemurafenib, in metastatic melanoma patients with the BRafV600E mutation. Approximately 50% of melanomas have an activating mutation to BRaf which drives cancer cell proliferation and survival through the ERK1/2 pathway. Therapies targeting BRaf or its downstream kinase MEK1/2 have demonstrated very promising initial clinical results. However, these treatments invariably lead to drug resistance through the activation of alternate pathways or mutations that circumvent the blockade and reactivate ERK1/2. Current efforts are centered on the use of combination therapies as well as the direct inhibition of ERK1/2 itself. The purpose of these studies is to characterize putative substrate-selective ERK inhibitors and examine their effects in various clinically relevant models of drug resistance. Specifically melanoma cell lines resistant to BRaf and MEK inhibitors, PLX-4032 and AZD-6244 respectively, were generated to test the efficacy of these novel compounds in cell viability assays. Furthermore, the potency of these molecules was evaluated in drug resistant cell lines that overexpress MAP3K8 (the gene encoding COT/Tpl2). The data show that these putative substrate-selective ERK inhibitors preferentially inhibit proliferation of cell lines with activated ERK1/2 signaling. In addition, these novel molecularly targeted therapies are equipotent in drug resistant versus non-resistant melanoma cells. Given these compounds' selectivity for BRaf mutated melanoma cells and their efficacy in drug resistance models, we submit these novel chemical entities as potential lead candidates for new salvage therapies in patients who have become resistant to currently available treatments. Moreover, these ATP-independent inhibitors target only select ERK1/2 functions and may be inherently less susceptible to developing their own drug resistance as compared to complete ablation of all ERK1/2 signaling as seen with ATP-competitive inhibitors.
    • Regulation of Class III Beta-Tubulin by Src-Mediated Tyrosine Phosphorylation

      Alfano, Alan; Qiu, Yun (2015)
      Prostate cancer (PCa) is the second most lethal cancer in men, accounting for an estimated 30,000 deaths in 2013. For diagnoses of advanced disease, the standard treatment is androgen deprivation therapy (ADT). Despite initial success of ADT, many PCa patients relapse into an incurable androgen-insensitive disease termed Castration Resistant Prostate Cancer (CRPC). Taxane compounds (such as docetaxel [DTX]) + Steroid (such as Prednisone) is a first-line treatment, but DTX resistance is common in patients with advanced CRPC. Tubulins are an integral part of the cytoskeleton, and play a pivotal role in cell signaling, migration, and division. They exist in vivo either as soluble monomers or as α-/β-tubulin heterodimers. These dimers are either recycled into monomers, or polymerized into microtubules (MTs). Microtubules serve a range of functions including structural support of the cell body, scaffolding for signaling molecules, and mitotic spindle support. β-tubulin is also the molecular target for taxane compounds. High expression levels of Class III β-tubulin (TUBB3, a primarily neural isoform of β-tubulin) correlate with taxane resistance and poor prognosis in several human cancers, including ovarian cancer (serous adenocarcinoma), breast cancer, non-small-cell lung cancer (NSCLC), and prostate cancer (PCa). It is known that c-Src (a known proto-oncogene) phosphorylates β-tubulins during both hematopoietic and neural differentiation. This has also been observed in leukemia cells. Despite recent advances, the relationship between Src-mediated tyrosine phosphorylation and microtubule modulation is still poorly understood, especially in the context of advanced solid malignancies. Herein we show that activated Src-kinase is able to phosphorylate TUBB3 at tyrosine 340 (Y340), and that tyrosine phosphorylation of βIII-tubulin at Y340 by Src-family kinases is critical in stabilization of TUBB3, and also plays a role in regulation of mitotic spindles. Given the clinical utility of TUBB3 as a biomarker of poor prognosis, characterizing cancer-specific post-translational modification (PTM) of TUBB3 in aggressive cancer cell types could lead to more specified biomarkers of patient outcome or therapeutic response. Additionally, a deeper understanding of the complex interplay between the Src family of kinases (SFKs) and microtubules could enable future researchers to design more specific and effective microtubule-targeted and/or SFK-targeted therapies.
    • Role of Nrf2 Signaling in Therapeutic Resistance and Cancer Metastasis

      Khatri, Raju; Jaiswal, Anil K. (2013)
      The stress response transcription factor Nrf2 is a master regulator of cytoprotective genes. In non-stress situation, INrf2 (Keap1), constantly sequesters Nrf2 and facilitates its ubiquitination-mediated proteasomal degradation. Upon exposure to stress, Nrf2 stabilizes, translocates to nucleus and binds to antioxidant response elements (ARE) in the promoter of its target genes and activates transcription of many cellular defense genes, resulting in cell survival and cytoprotection. Consequently, upregulation of Nrf2 results in development of therapeutic resistance. Aromatase inhibitors (AI) have been used for treatment of ERα positive breast cancer in post-menopausal women. However, persistent treatment with AI can lead to resistance. Little is known about the underlying mechanisms for the development of AI-resistance. In this study, we examined the involvement of Nrf2 signaling in AI-resistant cells and found downregulation of INrf2 and upregulation of Nrf2 and its target genes such as anti-apoptotic proteins, antioxidants and drug efflux transporters. Importantly, Nrf2- knock down AI-resistant cells showed fewer tumor-initiating cells (TIC), formed fewer mammospheres and become more sensitive to doxorubicin and etoposide. A stable cell line containing a luciferase reporter under the control of an ARE-promoter was generated to identify Nrf2 inhibitors by high-throughput screening to develop an adjuvant therapy that contains an Nrf2 inhibitor to enhance the therapeutic efficacy of aromatase inhibitors. We also investigated the role of Nrf2 in prostate cancer metastasis by generating stable cell lines expressing different levels of Nrf2. We investigated the role of Nrf2 signaling in colony formation, cell proliferation, migration and invasion. Our findings suggested that loss of Nrf2 leads to increased anchorage independent cell growth, proliferation, migration and invasion. In conclusion, persistent AI-treatment downregulated INrf2 leading to higher expression of Nrf2 and cytoprotective proteins that may, in part, result in increased AI-resistance. As knocking down Nrf2 decreased TIC, the inhibition of Nrf2 signaling appears to be beneficial to reduce AI-resistance. However, lowering the levels of Nrf2 might program cancer cells towards invasion and migration. Thus, understanding the levels of Nrf2 in tumors would be important to determine whether inhibition or activation of Nrf2 is beneficial to a cancer therapy.
    • Therapeutic Effect of Anti-Progranulin/GP88 Antibody AG01 in Triple Negative and Letrozole Resistant ER+ Breast Cancer Cells

      Guha, Rupa; Serrero, Ginette (2020)
      Progranulin (GP88, PCDGF, granulin/epithelin precursor, acrogranin) is a secreted autocrine growth/survival glycoprotein that functions as a biological driver of tumor cell proliferation, tumorigenesis, survival, invasiveness and drug resistance in several cancers, including breast cancer. Progranulin is found in the serum of breast cancer patients at higher levels than in healthy subjects and pathological studies have shown that in ER+ tumor biopsies, progranulin/GP88 is an independent prognostic factor of recurrence. Although TNBC represents a small percentage (15-20%) of breast cancer diagnoses, it is clinically important because of its highly aggressive nature and the fact that the disease progresses to metastasis within an exceedingly shorter period. Higher progranulin levels have also been shown to be associated with TNBC cases. Progranulin represents a therapeutic and diagnostic target in breast cancer. We have characterized a recombinant neutralizing anti-human progranulin/GP88 monoclonal antibody AG01 that inhibits progranulin biological effect in vitro and in vivo. Since GP88 is associated with poor outcomes in BC patients, we have investigated the effect of AG01 to inhibit proliferation and enhance letrozole responsiveness of letrozole resistance breast cancer cell lines as well as inhibit proliferation and migration of TNBC cells, two breast cancer areas with unmet medical needs for targeted therapy. We found that progranulin levels were sharply elevated in letrozole resistant cells as compared to the parent cell lines. Simultaneously, TNBC cells showed an increase in progranulin expression while it is undetectable in normal mammary cells. This emphasized the importance of targeting PGRN to treat letrozole resistance in ACLRTUSM as well as provide a therapeutic agent in TNBC cells. We report here that treatment of ACLRTUSM with anti-PGRN antibody (AG01) not only reduced their proliferation but increased the sensitivity of ACLRTUSM cells towards letrozole treatment. In several TNBC models, AG01 treatment reduced cell proliferation, migration, and invasion. Taken together, the research work discussed here provides new information to better understand the targeting progranulin and the effectiveness of AG01 as a potential therapeutic agent in breast cancer. Future work continuing characterization of AG01 will provide further insight into its role in regulating cancer biology.