Wnt/β-Catenin mediated cancer stem cell activation in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers, with variable presentation, high chemotherapy resistance and early tumor recurrence. In this dissertation, we have systematically enriched, identified and characterized HCC cancer stem cells (CSCs) from 3 different HCC cell lines (Hepa1-6, HepG2, and Hep3B) using in vitro serum-free culture method. Enriched spheroids acquired CSC properties, primarily self-renewal capability, spheroid formation ability, and drug resistance. Heterogeneous CSCs enriched from Hepa1-6 cells demonstrated higher tumor initiation and proliferation capability in vivo, compared to control HCC cells in orthotopic immunocompetent mouse model. EpCAM+ CSCs were significantly enriched within heterogeneous Hepa1-6 CSC spheroids. Consequently, we studied the fate of EpCAM+ CSCs in orthotopic immunocompetent mouse model with 3 different liver microenvironments i.e. control normal liver, steatosis (fatty liver induced), and nonalcoholic steatohepatitis (NASH). EpCAM+ CSC mediated HCC carcinogenesis was observed in NASH livers, but failed to develop in control and steatosis liver microenvironment. This is the first study evaluating CSCs in immunocompetent mouse model and demonstrated the importance of liver microenvironment for EpCAM+ CSC mediated tumor initiation. We then performed a lineage tracking study by stably incorporating copGFP or mCherry in Hepa1-6 cells by lentivirus transduction, and examined the origin of tumors in NASH liver microenvironment using orthotopic C57L/J immunocompetent mouse model. Our findings suggest that tumor growth was dose dependent and most tumor nodules arose from copGFP expressing EpCAM+ CSCs. The Wnt/β-catenin pathway components were also found to be overexpressed in CSCs when compared to control. Human HCC specimen analyses suggested concomitant changes in β-catenin and EpCAM levels. Gene analyses of step-wise spheroid formation process identified possible dedifferentiation mechanisms regulated by β-catenin in CSC spheroids. Loss of function analysis by siRNA mediated transient knockdown of β-catenin confirmed its role in spheroid enrichment and doxorubicin resistance. Canonical Wnt pathway study using chemical inhibitors identified β-catenin mediated CSC activation was regulated at nuclear level, and not at the cytoplasmic GSK3β destruction complex level. Analysis of human and mouse tumors suggested that constitutive activation of β-catenin transcription failed to respond to NOTUM, a feedback inhibitor of canonical Wnt/β-catenin signaling

The Promise of Sonodynamic Therapy: Using Ultrasonic Irradiation and Chemotherapeutic Agents as a Treatment Modality

Sonodynamic therapy (SDT) is a potential cancer treatment modality that has been gaining support due to its effectiveness in both in vitro and in vivo studies. The therapeutic method combines ultrasonic irradiation with drugs known as sonosensitizers that amplify its ability to inflict preferential damage on malignant cells. This is based on the idea that ultrasonic waves have the ability to exhibit profound physical and chemical changes on cellular structure. The mechanisms by which ultrasound disrupts cellular functioning can be further amplified when sonosensitizers are applied. Combining multiple sonosensitizers with ultrasound to create a substantial synergistic effect could be an effective method for destroying tumorigenic growths, while decreasing the likelihood of drug resistance. Perhaps one of the most intriguing capabilities of ultrasound is its ability to preferentially lyse cells based on size. This known fact invariably gives rise to the idea of grossly enlarging tumor cells to increase their already noticeable size difference with normal cells. Cytochalasin B is a known pharmacological agent that disrupts the actin cytoskeleton and inhibits cytokinesis by interfering with formation of the contractile ring as well as the development of the cleavage furrow. Consequently, the cell does not divide and an immature actin cytoskeleton remains. However, the cell continues to form nuclei and eventually becomes grossly enlarged and multinucleated. Such cells invariably have more DNA targets, increasing the likelihood of apoptosis. Furthermore, the multinucleated cells have a large cell volume, making them more susceptible for direct cell destruction. Preferential damage of malignant cells is actually easily attainable as normal cells exposed to cytochalasin B exit the cell cycle and enter a resting state until sufficient actin levels are restored. Therefore, only malignant cells that have lost the ability to enter the rest phase will become grossly enlarged and multinucleated, providing an ideal target for ultrasonic irradiation. Work from our lab has indicated that cytochalasin B does indeed only damage leukemia cells, leaving normal blood cells, unaffected. The designated cell line has been promyleocytic leukemia U937 cells as they are a frequent choice for in vitro studies. The U937 cells have routinely become grossly enlarged and multinucleated, providing an ideal target based on size. The typical erythrocyte is 6-8µm, while leukocytes fair slightly better with a range of 10-15µm and an average of 12µm. By contrast, work from our lab has shown that cytochalasin B treated leukemia cells easily grow in excess of 20µm with some reaching 40µm in diameter after enough exposure. Such cells have reduced cytoskeletal integrity and are easy targets for ultrasonic irradiation. Furthermore, cytochalasin B treated leukemia cells are substantially multinucleated as cytokinesis is inhibited. This provides plenty of targets for a nucleic acid directed agent such as cisplatin or doxorubicin to attack. To investigate the extent of preferential damage inflicted by cytochalasin B on U937 leukemia/human blood populations, cell mixtures were treated with cytochalasin B and then sonicated under a relatively low intensity (3W/cm2). Results indicated that cytochalasin B preferentially damages U937 cells both before and after sonications. The agent also demonstrates the capability to eliminate rapid proliferation as U937 cells have a marked decrease in clonogenicity. Such findings suggest that cytochalasin B may have profound therapeutic applications when combined with SDT. Key Words Sonodynamic Therapy, Ultrasound, Sonosensitizers, Inertial Cavitation, Reactive Oxygen Species, Tumor Vasculature, Preferential Damag

Hepatocellular carcinoma and multidrug resistance: Past, present and new challenges for therapy improvement

Hepatocellular carcinoma (HCC) is the most common malignancy of the liver and the third cause of cancer death worldwide. Chronic hepatitis due to HBV and HCV infection are two major risk factors for HCC worldwide. Advances in early detection and treatment have improved life expectancy of patients with HCC. However, this disorder remains as a disease with poor prognosis. In fact, epidemiological studies have shown that the median survival of patients is 8 months and approximately 20% of them survive one year, while only 5% remain alive after three years. Additionally, HCC is particularly difficult to treat because of its high recurrence rate, and its resistance to conventional chemotherapy due to, among other mechanisms, the over-expression of several members of the ATP-Binding Cassette (ABC) protein family involved in drugs transport. Fortunately, there is evidence that these patients may benefit from alternative molecular-targeted therapies. This manuscript reviews the current knowledges on the etiology, molecular mechanisms involved in HCC development and the current therapy strategies for the management of this malignancy. The challenges in the development of drug delivery systems for the targeting of antitumoral drugs to the liver parenchyma are also discussed. Finally, perspectives in the design of a more efficient pharmacotherapy to overcome multidrug resistance are reviewed.Fil: Cuestas, María Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Oubiña, Jose Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Mathet, Veronica Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentin

Cancer Therapy Combining Modalities of Hyperthermia and Chemotherapy: in vitro Cellular Response after Rapid Heat Accumulation in the Cancer Cell

Hyperthermia is usually used at a sub-lethal level in cancer treatment to potentiate the effects of chemotherapy. The purpose of this study is to investigate the role of heating rate in achieving synergistic cell killing by chemotherapy and hyperthermia. For this purpose, in vitro cell culture experiments with a uterine cancer cell line (MES-SA) and its multidrug resistant (MDR) variant MES-SA/Dx5 were conducted. The cytotoxicitiy, mode of cell death, induction of thermal tolerance and P-gp mediated MDR following the two different modes of heating were studied. Doxorubicin (DOX) was used as the chemotherapy drug. Indocyanine green (ICG), which absorbs near infrared light at 808nm (ideal for tissue penetration), was chosen for achieving rapid rate hyperthermia. A slow rate hyperthermia was provided by a cell culture incubator. The results show that the potentiating effect of hyperthermia to chemotherapy can be maximized by increasing the rate of heating as evident by the results from the cytotoxicity assay. When delivered at the same thermal dose, a rapid increase in temperature from 37 °C to 43 °C caused more cell membrane damage than gradually heating the cells from 37 °C to 43 °C and thus allowed for more intracellular accumulation of the chemotherapeutic agents. Different modes of cell death are observed by the two hyperthermia delivery methods. The rapid rate laser-ICG hyperthermia @ 43 °C caused cell necrosis whereas the slow rate incubator hyperthermia @ 43 °C induced very mild apoptosis. At 43 °C a positive correlation between thermal tolerance and the length of hyperthermia exposure is identified. This study shows that by increasing the rate of heating, less thermal dose is needed in order to overcome P-gp mediated MDR

The Role of FOXO3 in Mechanisms of Doxorubicin Resistance in Hepatocellular Carcinoma

Trans-arterial chemoembolization (TACE) with doxorubicin is commonly used to treat hepatocellular carcinoma (HCC), but has limited efficacy due to a high level of resistance. The factors that determine the sensitivity to TACE-doxorubicin are unknown. FOXO3 is a multifunctional transcription factor that plays a role in determining cell fate in response to stress. It frequently functions as a tumor suppressor but can also promote tumor pathogenesis. FOXO3 is also known to be a mediator of doxorubicin sensitivity in many types of tumor cells, while in others it can promote resistance. The role of FOXO3 in HCC and in doxorubicin resistance in HCC is unknown. FOXO3 function is largely determined by post-translational modification (PTM). Two FOXO3 PTMs, acetylation and serine 574 (S574)-phosphorylation, are known to promote its apoptotic function. Contrary to expectations, expression of FOXO3 was increased in HCC compared to surrounding liver. Cytosolic FOXO3 was significantly greater in TACE-resistant HCC as compared to treatment-naïve tumors. In Huh7 hepatoma cells, doxorubicin induced acetylation and S574-phosphorylation of FOXO3, and these modifications promoted doxorubicin-induced cell death by suppressing the pro-survival function of FOXO3. Resveratrol, an activator of SIRT deacetylase enzymes, inhibited these doxorubicin-induced PTMs and increased doxorubicin resistance. The expression of SIRT6, a known FOXO3 deacetylase, was also increased in TACE-resistant HCC tumors and correlated with cytosolic FOXO3. SIRT6 also blocked doxorubicin-induced S574-phosphorylation of FOXO3 and increased resistance to doxorubicin in Huh7 cells. Therefore, targeting SIRT6 and/or manipulating FOXO3 modifications may prove useful in enhancing the chemotherapy sensitivity of HCC

Doxorubicin and other anthracyclines in cancers: activity, chemoresistance and its overcoming

Anthracyclines have been important and effective treatments against a number of cancers since their discovery. However, their use in therapy has been complicated by severe side effects and toxicity that occur during or after treatment, including cardiotoxicity. The mode of action of anthracyclines is complex, with several mechanisms proposed. It is possible that their high toxicity is due to the large set of processes involved in anthracycline action. The development of resistance is a major barrier to successful treatment when using anthracyclines. This resistance is based on a series of mechanisms that have been studied and addressed in recent years. This work provides an overview of the anthracyclines used in cancer therapy. It discusses their mechanisms of activity, toxicity, and chemoresistance, as well as the approaches used to improve their activity, decrease their toxicity, and overcome resistance

Doctor of Philosophy

dissertationThermal ablation is widely used, first line local-regional therapy for unresectable hepatocellular carcinoma (HCC). Although high temperature delivered by thermal energy results in efficient coagulation necrosis in tumor cells, various factors including tumor size, shape, location, and cirrhosis can lead to un-uniform heat distribution and inefficient cell damage. As a result, the incomplete ablation causes high rates of tumor recurrence and poor survival for HCC patients. Cells that are not completely ablated can induce heat shock proteins (HSPs), which are cellular gatekeepers to protect tumor cells from thermal damage and prepare them for future neoplastic growth. Synchronous adjuvant chemotherapy targeting those cells can achieve more complete tumor abrogation and prevent future tumor recurrence. This dissertation describes a strategy to combat postablation recurrence by synchronous inhibition of heat shock protein 90 (HSP90) by thermo-responsive, elastin-like polypeptide (ELP)-based biopolymer conjugates. ELP copolymer carries high concentrations of a potent HSP90 inhibitor, geldanamycin (GA), which inhibit the induction of HSP90 and further destabilize numerous HSP90 client proteins critical for cell survival. It is hypothesized that combination of thermal ablation with concomitant inhibition of HSP90 via ELP-GA conjugates can achieve synergistic anticancer effect. Specifically, the ablation-created hyperthermia will sensitize tumor cells to be more vulnerable to the drug, which will be conjugated with high concentrations through thermally targeted, ELP-based biopolymer systems. The ELP conjugates, in turn, will reach and kill the remaining viable cells to prevent future recurrence. ELP-GA conjugates that ferry multiple GAs and rapidly respond to hyperthermia were synthesized, characterized, and evaluated for activity in HCC models. The cytotoxicity of ELP-GA conjugates was enhanced with hyperthermia treatment, and effective HSP90 inhibition was achieved in HCC cell lines. In a tumor-bearing mouse model, electrocautery-based thermal ablation offered effective destruction of tumor core and created a hyperthermia zone for targeted delivery and accumulation of ELP-GA conjugates. Results demonstrate that the combination of thermal ablation and targeted HSP90 inhibition can enhance the anticancer effect and cellular delivery of macromolecular chemotherapeutics to achieve safe, synergistic, and long-term anticancer effect with no tumor recurrence observed. The combination approach paves the way for developing molecular-targeted intervention to increase the efficacy of first-line local-regional therapies for HCC