To Divide or Not to Divide; MicroRNAs and Small Compounds as Modulators of Mitosis

Mitosis is under the stringent quality control of the spindle assembly checkpoint (SAC). However, in cancer cells this control can fail, leading to excessive cellular proliferation and ultimately to the formation of a tumor. Novel cancer cell selective therapies are needed to stop the uncontrolled cell proliferation and tumor growth. The aim of the research presented in this thesis was to identify microRNAs (miRNAs) that could play a role in cancer cell proliferation as well as low molecular weight (LMW) compounds that could interfere with cell division. The findings could be used to develop better cancer diagnostics and therapies in the future. First, a high-throughput screen (HTS) was performed to identify LMW compounds that possess a similar chemical interaction field as rigosertib, an anti-cancer compound undergoing clinical trials. A compound termed Centmitor-1 was discovered that phenocopied the cellular impact of rigosertib by affecting the microtubule dynamics. Next, another HTS aimed at identifying compounds that would target the Hec1 protein, which mediates the interaction between spindle microtubules and chromosomes. Perturbation of this connection should prevent cell division and induce cell death. A compound termed VTT-006 was discovered that abrogated mitosis in several cell line models and exhibited binding to Hec1 in vitro. Lastly, using a cell-based HTS two miRNAs were identified that affected cancer cell proliferation via Aurora B kinase, which is an important mitotic regulator. MiR-378a-5p was found to indirectly suppress the production of the kinase whereas let-7b showed direct binding to the 3’UTR of Aurora B mRNA and repressed its translation. The miRNA-mediated perturbation of Aurora B induced defects in mitosis leading to abnormal chromosome segregation and induction of aneuploidy. The results of this thesis provide new information on miRNA signaling in cancer, which could be utilized for diagnostic purposes. Moreover, the thesis introduces two small compounds that may benefit future drug research.Siirretty Doriast

Optimizing differentiation of human pluripotent stem cells towards corneal epithelium

Background and aims: Limbal stem cell deficiency (LSCD) is a potentially blinding corneal disease caused by trauma, such as chemical burn, inflammation, or hereditary factors. LSCD is characterized by dysfunction of limbal epithelial stem cells (LESC) and failure of barrier function of limbus. Surrounding conjunctival tissue invades transparent and avascular corneal epithelium, which leads to vascularization and haziness. In LSCD, the surface of the eye cannot recover itself due to lack or dysfunction of LESCs so the treatment needs to bring new stem cells to continue tissue regeneration. Various approaches have been proposed to treat LSCD, some using limbal tissue transplantations from healthy eye and some have been cell-based therapies. This study presents a method developed in the Eye group at the University of Tampere, which uses human pluripotent stem cells (hPSCs) to differentiate corneal epithelial-like cells. This study's aim was to further optimize this method and test enzymatic dissociation method for stem cells. Methods: In this study, cell differentiation was repeated six times. In every experiment, hPSCs were dissociated from feeder cells either mechanically or enzymatically. The cells were suspension cultured in small molecule and growth factor supplemented induction medium as embryoid bodies (EBs). After seven-day induction, the cells were plated to well plates, which were coated with collagen IV or surface x. The adherent cell culture lasted 21 days. Cells were analyzed with immunofluorescence at midpoint and end point of the experiments, to identify either LESC-like cells or corneal epithelial cells. Results: Mechanical and enzymatic dissociation methods both worked as detaching methods. Enzymatic dissociation gave partially better cell numbers than mechanical dissociation. EBs survived better in suspension culture when the growth area was smaller. Collagen IV and surface x both worked as growth surface but cell numbers were better with surface x. In adherent cell culture, the growth area proved to be important, because cells seemed to suffer from overcrowding. At the end point of the experiments, there were cells positive for both LESC and cornea epithelium markers. Conclusions: Enzymatic dissociation method worked as a dissociation method alongside the mechanical dissociation. Enzymatically dissociated EBs survived and outgrowths were better than mechanically dissociated EBs. Surface x improved cell attachment and enhanced their differentiation towards corneal epithelium

Additive Benefits of Radium-223 Dichloride and Bortezomib Combination in a Systemic Multiple Myeloma Mouse Model

Osteolytic bone disease is a hallmark of multiple myeloma (MM) mediated by MM cell proliferation, increased osteoclast activity, and suppressed osteoblast function. The proteasome inhibitor bortezomib targets MM cells and improves bone health in MM patients. Radium-223 dichloride (radium-223), the first targeted alpha therapy approved, specifically targets bone metastases, where it disrupts the activity of both tumor cells and tumor-supporting bone cells in mouse models of breast and prostate cancer bone metastasis. We hypothesized that radium-223 and bortezomib combination treatment would have additive effects on MM. In vitro experiments revealed that the combination treatment inhibited MM cell proliferation and demonstrated additive efficacy. In the systemic, syngeneic 5TGM1 mouse MM model, both bortezomib and radium-223 decreased the osteolytic lesion area, and their combination was more effective than either monotherapy alone. Bortezomib decreased the number of osteoclasts at the tumor-bone interface, and the combination therapy resulted in almost complete eradication of osteoclasts. Furthermore, the combination therapy improved the incorporation of radium-223 into MM-bearing bone. Importantly, the combination therapy decreased tumor burden and restored body weights in MM mice. These results suggest that the combination of radium-223 with bortezomib could constitute a novel, effective therapy for MM and, in particular, myeloma bone disease

VTT-006, an anti-mitotic compound, binds to the Ndc80 complex and suppresses cancer cell growth <i>in vitro</i>.

Hec1 (Highly expressed in cancer 1) resides in the outer kinetochore where it works to facilitate proper kinetochore-microtubule interactions during mitosis. Hec1 is overexpressed in various cancers and its expression shows correlation with high tumour grade and poor patient prognosis. Chemical perturbation of Hec1 is anticipated to impair kinetochore-microtubule binding, activate the spindle assembly checkpoint (spindle checkpoint) and thereby suppress cell proliferation. In this study, we performed high-throughput screen to identify novel small molecules that target the Hec1 calponin homology domain (CHD), which is needed for normal microtubule attachments. 4 million compounds were first virtually fitted against the CHD, and the best hit molecules were evaluated in vitro. These approaches led to the identification of VTT-006, a 1,2-disubstituted-tetrahydro-beta-carboline derivative, which showed binding to recombinant Ndc80 complex and modulated Hec1 association with microtubules in vitro. VTT-006 treatment resulted in chromosome congression defects, reduced chromosome oscillations and induced loss of inter-kinetochore tension. Cells remained arrested in mitosis with an active spindle checkpoint for several hours before undergoing cell death. VTT-006 suppressed the growth of several cancer cell lines and enhanced the sensitivity of HeLa cells to Taxol. Our findings propose that VTT-006 is a potential anti-mitotic compound that disrupts M phase, impairs kinetochore-microtubule interactions, and activates the spindle checkpoint

Effect of remdesivir post hospitalization for COVID-19 infection from the randomized SOLIDARITY Finland trial

We report the first long-term follow-up of a randomized trial (NCT04978259) addressing the effects of remdesivir on recovery (primary outcome) and other patient-important outcomes one year after hospitalization resulting from COVID-19. Of the 208 patients recruited from 11 Finnish hospitals, 198 survived, of whom 181 (92%) completed follow-up. At one year, self-reported recovery occurred in 85% in remdesivir and 86% in standard of care (SoC) (RR 0.94, 95% CI 0.47-1.90). We infer no convincing difference between remdesivir and SoC in quality of life or symptom outcomes (p > 0.05). Of the 21 potential long-COVID symptoms, patients reported moderate/major bother from fatigue (26%), joint pain (22%), and problems with memory (19%) and attention/concentration (18%). In conclusion, after a one-year follow-up of hospitalized patients, one in six reported they had not recovered well from COVID-19. Our results provide no convincing evidence of remdesivir benefit, but wide confidence intervals included possible benefit and harm.Peer reviewe

Optimizing differentiation of human pluripotent stem cells towards corneal epithelium

Background and aims: Limbal stem cell deficiency (LSCD) is a potentially blinding corneal disease caused by trauma, such as chemical burn, inflammation, or hereditary factors. LSCD is characterized by dysfunction of limbal epithelial stem cells (LESC) and failure of barrier function of limbus. Surrounding conjunctival tissue invades transparent and avascular corneal epithelium, which leads to vascularization and haziness. In LSCD, the surface of the eye cannot recover itself due to lack or dysfunction of LESCs so the treatment needs to bring new stem cells to continue tissue regeneration. Various approaches have been proposed to treat LSCD, some using limbal tissue transplantations from healthy eye and some have been cell-based therapies. This study presents a method developed in the Eye group at the University of Tampere, which uses human pluripotent stem cells (hPSCs) to differentiate corneal epithelial-like cells. This study's aim was to further optimize this method and test enzymatic dissociation method for stem cells. Methods: In this study, cell differentiation was repeated six times. In every experiment, hPSCs were dissociated from feeder cells either mechanically or enzymatically. The cells were suspension cultured in small molecule and growth factor supplemented induction medium as embryoid bodies (EBs). After seven-day induction, the cells were plated to well plates, which were coated with collagen IV or surface x. The adherent cell culture lasted 21 days. Cells were analyzed with immunofluorescence at midpoint and end point of the experiments, to identify either LESC-like cells or corneal epithelial cells. Results: Mechanical and enzymatic dissociation methods both worked as detaching methods. Enzymatic dissociation gave partially better cell numbers than mechanical dissociation. EBs survived better in suspension culture when the growth area was smaller. Collagen IV and surface x both worked as growth surface but cell numbers were better with surface x. In adherent cell culture, the growth area proved to be important, because cells seemed to suffer from overcrowding. At the end point of the experiments, there were cells positive for both LESC and cornea epithelium markers. Conclusions: Enzymatic dissociation method worked as a dissociation method alongside the mechanical dissociation. Enzymatically dissociated EBs survived and outgrowths were better than mechanically dissociated EBs. Surface x improved cell attachment and enhanced their differentiation towards corneal epithelium

Human Immune System Increases Breast Cancer-Induced Osteoblastic Bone Growth in a Humanized Mouse Model without Affecting Normal Bone

Bone metastases are prevalent in many common cancers such as breast, prostate, and lung cancers, and novel therapies for treating bone metastases are needed. Human immune system-engrafted models are used in immuno-oncology (IO) studies for subcutaneous cancer cell or patient-derived xenograft implantations that mimic primary tumor growth. Novel efficacy models for IO compounds on bone metastases need to be established. The study was performed using CIEA NOG (NOG) mice engrafted with human CD34+ hematopoietic stem cells (huNOG) and age-matched immunodeficient NOG mice. Bone phenotyping was performed to evaluate baseline differences. BT-474 human breast cancer cells were inoculated into the tibia bone marrow, and cancer-induced bone changes were monitored by X-ray imaging. Bone content and volume were analyzed by dual X-ray absorptiometry and microcomputed tomography. Tumor-infiltrating lymphocytes (TILs) and the expression of immune checkpoint markers were analyzed by immunohistochemistry. Bone phenotyping showed no differences in bone architecture or volume of the healthy bones in huNOG and NOG mice, but the bone marrow fat was absent in huNOG mice. Fibrotic areas were observed in the bone marrow of some huNOG mice. BT-474 tumors induced osteoblastic bone growth. Bone lesions appeared earlier and were larger, and bone mineral density was higher in huNOG mice. huNOG mice had a high number of human CD3-, CD4-, and CD8-positive T cells and CD20-positive B cells in immune-related organs. A low number of TILs and PD-1-positive cells and low PD-L1 expression were observed in the BT-474 tumors at the endpoint. This study reports characterization of the first breast cancer bone growth model in huNOG mice. BT-474 tumors represent a “cold” tumor with a low number of TILs. This model can be used for evaluating the efficacy of combination treatments of IO therapies with immune-stimulatory compounds or therapeutic approaches on bone metastatic breast cancer