The Reelin gene located on the human chromosome region 7q22, encodes an extracellular matrix glycoprotein, a ligand for ApoER2 and low-density lipoprotein receptors (LDL) Receptor, is required for mediating the correct positioning of neurons during embryonic brain development1. In the current study, first we applied RT-PCR and immunohistochemistry analysis (IHC) analysis on tissue microarrays (TMA) to verify the Reelin expression patterns in a variety of adult tissues, suggesting additional roles for Reelin in stabling the cyto-architecture and controlling the remodeling of many organs during development. Second, we report the Reelin expression status in tumorigenesis. We discover that the loss of Reelin expression is associated with multiple types of cancers, including more than 80% of both breast and colorectal cancers. Interestingly, our study also found suspension small cell lung cancer (SCLC) cell lines that grow as large aggregates retained high Reelin expression, whereas attached non small cell lung cancer cultures do not. That may imply the Reelin expression may be also associated with cell culture morphology and growth characteristics in the in vitro culture system for lung cancers. Our results here also demonstrated that epigenetic silencing of Reelin expression by DNA hypermethylation in tumors directly correlates with loss of Reelin expression in many cancers. Reelinmethylation was reversed and expression restored by treating tumor cell lines with the demethylating agent 5-aza-2-deoxycytidine. In conclusion, from the molecular basis of Reelingene inactivation in human cancer here, we propose that the Reelinvariation in more than 80% of breast and colorectal cancers makes it a significant novel tumor marker.

Small cell lung cancer (SCLC) is characterized by prompt response to chemotherapy and radiotherapy but relapsing with drug resistance and distant metastasis, leading to poor overall prognosis. New anticancer agents and regimens are drastically needed for SCLC treatment. Arsenic trioxide (ATO), a traditional Chinese medicine used as a poison for thousands of years, has been tested in many hematological and solid cancers both in vitro and in vivo, with promising effects. In order to establish the scientific ground for future clinical application of ATO in SCLC, this study aimed to investigate the anticancer effect and mechanism of ATO in SCLC using in vitro and in vivo models, either as a single agent or in combination with standard chemotherapy. In addition, an ATO-acquired resistant cell line (H841-AR) derived from SCLC cell line H841 was used to explore potential mechanisms of ATO resistance and cross-resistance to other chemotherapeutic drugs. In the first part of this study, ATO was shown to exert cytotoxic effect in all of the chosen SCLC cell lines. Various cellular mechanisms were triggered upon ATO exposure: redox status disturbances (hydrogen peroxide (H2O2) generation, glutathione (GSH) depletion and thioredoxin 1 (Trx1) down-regulation), mitochondrial membrane depolarization (MMD), DNA damage, apoptosis and necroptosis. In concert with this, Bcl-2 was down-regulated accompanied by MMD, release of AIF and SMAC, DNA degradation, XIAP inhibition and caspases activation. Adoption of N-acetyl-L-cysteine (NAC) and buthionine sulfoximine (BSO) demonstrated GSH depletion and reactive oxygen species (ROS) generation played the pivotal role to mediate cytotoxic effect of ATO in SCLC. In the second part of this study, when combined with chemotherapeutic agents, ATO displayed synergistic and antagonistic interaction with cisplatin and etoposide respectively in SCLC cell line models. The beneficial combination of ATO and cisplatin was also substantiated by tumor xenograft models. Augmented GSH depletion and suppressed drug efflux mechanism were found to explain the synergistic effects. In the last part of this study, H841-AR was generated as an acquired multi-drug resistant (ATO, cisplatin and etoposide) cell line to investigate the potential resistance mechanisms and possible future drug combinations. Comparing H841-AR cells with parental H841 cells using cDNA microarray, a long list of genes was altered in ATO-resistant cells. At least 20 up-regulated and 45 down-regulated genes were short-listed as candidates with a cut-off at 5-fold change. Interestingly, qPCR data has shown that 5 selected up-regulated genes in H841-AR cells were also highly expressed in DMS79 cells with intrinsic ATO resistance compared to the relatively sensitive cell lines, indicating that these genes might be associated with ATO resistance in SCLC. In summary, ATO was shown to be an active anticancer agent in SCLC, either alone or in combination with cisplatin. The major mechanisms of action of ATO and its synergism with cisplatin in SCLC were elucidated. Genetic data derived from an acquired resistant (to ATO, cisplatin and etoposide) SCLC cell line may help to uncover the mechanisms of resistance to ATO, allowing possible future drug combinations. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumour which secretes ACTH and other related peptides. Contrary to normal production by the pituitary, ACTH production is not inhibited by glucocorticoids (Gcs) in SCLC. This insensitivity to Gc action can be attributed to impaired Gc receptor (GR) expression in these cells. Over-expression of the GR induces apoptosis both in vitro and in vivo. Evasion of GR signalling thus confers a significant survival advantage to SCLC cells. Re-expression of endogenous GR in SCLC cells may provoke the same effect. Many tumours silence the expression of tumour suppresser genes by epigenetic mechanisms. Recent evidence suggests that the GR in SCLC cells is epigenetically silenced by hypermethylation of its promoter. The overall aim of this study was to determine whether endogenous GR re-expression induces apoptosis of SCLC cells. The DMS 79 SCLC cell line, and the control HEK and non-SCLC A549 cell lines were treated with the DNA methyltransferase inhibitor (DNMTi), 5-aza-2′-deoxycytidine (5-aza), to determine whether treatment with 5-aza results in re-expression of endogenous GR. Conflicting results were thought to result from the use of possibly degraded 5-aza. However, a quantitative real-time PCR analysis using newly purchased, freshly prepared 5-aza indicated that 5-aza treatment up-regulated GR mRNA expression in the DMS 79 cells (p<0.0005). No significant changes in GR expression were seen in the HEK and/or A549 cells, suggesting that the GR in these cell lines is not methylated. Contrary to expectations and possibly due to the use of degraded stock, Western blot analysis revealed that 5-aza had no effect on GR protein expression in DMS 79 cells, yet affected GR protein expression in HEK and A549 cells (p=0.003 and p=0.042, respectively). Cell viability assays indicated that treatment with varying concentrations of 5-aza had no effect on the viability of DMS 79 and A549 cells, but had a minimal effect on HEK cell (p<0.0005) viability. These data reinforce the hypothesis that stock 5-aza had degraded as 5-aza is known to exert cytotoxic effects at higher concentrations. Using newly purchased, freshly prepared 5-aza, flow cytometry and/or microscopy were performed to establish whether endogenous GR re-expression was sufficient to kill the SCLC cells by apoptosis. FITC Annexin V staining and nuclear morphology showed that significant proportions of the 1 μM (p=0.010 and p=0.027) and 5 μM (p=0.002 and p=0.018) 5-aza treated DMS 79 cells were apoptosing, with little apoptosis seen in HEK cells. 5-Aza induced negligible HEK cell death, as determined by microscopic analyses. The effect of dexamethasone (Dex; a synthetic Gc) on HEK and DMS 79 cells was examined to determine whether Gc treatment could enhance apoptosis. Treatment with Dex alone, and in combination with 5-aza, resulted in significant HEK cell death (p=0.046 and p=0.005 respectively), but not apoptosis. This was unexpected as HEK cells express very little unmethylated GR, and may be due to excessive drug exposure or combined drug toxicity. The same effect was observed with DMS 79 cells (p=0.003 and p<0.0005 respectively), with 5-aza appearing to enhance cell death induced by Dex. No effects on apoptosis were seen confirming earlier reports that GR-mediated apoptosis is ligand-independent. As 5-aza does not selectively demethylate the GR, cells were exposed to the GR antagonist, RU486, to establish whether apoptosis associated with 5-aza treatment is specifically due to demethylation and subsequent expression of the GR. Treatment with RU486 in conjunction with 5-aza induced cell death (p=0.014), but not apoptosis, of HEK cells. Again, this may have been due to excessive drug exposure or combined drug toxicity. Flow cytometric data showed that DMS 79 cell death was induced by both RU486 (p=0.004), and RU486 in combination with 5-aza (p=0.003). Furthermore, although not significant, RU486 treatment appeared to inhibit apoptosis induced by 5-aza in the DMS 79 cells. The data suggest that re-expression of the GR may be responsible for apoptotic induction. Our findings, although not significant, hint that endogenous re-expression of the GR leads to apoptosis. Unlike mutations, epigenetic marks are reversible and clinical trials with DNMTis have shown promising results. The identification of a novel endogenous mechanism that specifically induces apoptosis of SCLC cells offers great promise for the development of targeted therapeutics for the treatment of this deadly disease. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

Small cell lung cancer (SCLC) is an aggressive form of lung cancer that frequently develops multidrug resistance (MDR) during chemotherapy. Vault RNA1 (vRNA1), a non-structural component of the MDR-associated vault organelle, is believed to act as a microRNA (miRNA) and may contribute to MDR by regulating the expression of genes involved in apoptosis, inflammation, and/or drug metabolism. Since vaults function to aid cells in survival, we hypothesized that vRNA1 might be free in the cytoplasm and able to inhibit expression of pro-survival mRNAs when vaults are open in drug-sensitive GLC4/S cells but not in the MDR GLC4/ADR cells where vaults might be closed with the miRNA sequestered within. In order to establish the role of vRNA1 as a regulator of survival in SCLC cells, siRNA-mediated down-regulation of vRNA1 was employed in GLC4/S and GLC4/ADR SCLC cells. Fluorescence microscopy using a green fluorescent 3’ AlexaFluor-488 negative siRNA control was used to estimate transfection efficiency, yielding 56% for GLC4/S and 89% for GLC4/ADR. However, these values and the level of apoptosis before and after transfection, as judged by trypan blue hemacytometer cell counts, were not entirely reliable due to cell clumping. The latter counts indicated a 2-fold decrease in viability in GLC4/S cell following transfection but no decrease in GLC4/ADR cells (p< 0.05). RT-PCR revealed that transfection significantly (p<0.05) decreased vRNA1 expression in GLC4/S cells but not in GLC4/ADR cells, confirming our hypothesis concerning the availability of vRNA1 in the two cell types. Caspase activity measurements showed vRNA1 down-regulation in the GLC4/ADR cells significantly (p≤0.05) increased survival via a 6.1-fold reduction in caspase 3/7 activity, further supporting our hypothesis. However, GLC4/S cells showed a similar loss of apoptosis when transfected with either sivRNA1 or the negative control siRNA. vRNA1 down-regulation did not significantly (p≤0.05) affect the expression of major pro-survival (Bcl-2, Bcl-xL), pro-apoptotic (Bad), or pro-inflammatory (IL-6, NFĸB p65) factors in either GLC4/S or GLC4/ADR cells. However, the drug metabolism protein CYP3A (previously shown by Persson et al., 2009 to be regulated by vRNA1) was significantly (p≤0.05) lowered (~16%) following vRNA1 down-regulation in the GLC4/S cells. In conclusion, we were successful in down-regulating vRNA1 which enhanced cell survival as hypothesized, but we were not able to identify new proteins regulated by vRNA1. / Department of Biology

Only discovered about 20 years ago, the structure of the eukaryotic vault particle has been studied extensively, but the function has yet to be determined. Vault numbers are up regulated in many types of cancer cells that are treated with chemotherapy agents and it is thought that they may act to transport chemotherapy drugs out of such cells, leading to multi-drug resistance (MDR). To determine a possible role of the vault particle in MDR, the goal of this research was to examine one of the functional vault proteins, vault poly(ADP)ribose (VPARP) for interactions with other proteins. Two forms of small cell lung cancer cells were used; GLC4/S which do not exhibit MDR and the MDR cells GLC4/ADR, which are cultured with the chemotherapy drug doxorubicin. Both cell cultures were subjected to a subcellular fractionation followed by gentle immunoprecipitation with an antibody to VPARP. Immunoprecipitated proteins interacting with VPARP were only observed in GLC4/ADR cells, as seen on a PAGE gel. This sample was taken to Monarch Life Sciences and analyzed by mass spectrometry. One interacting protein was found to be NALP1 pyrin domain (PYD), a member of the death domain family of proteins which is involved in inflammation and apoptosis. The interaction of VPARP with NALP1, which only occurred in MDR cells, suggests an exciting, previously unreported possibility – that VPARP binding may inhibit NALP 1-stimulated apoptosis when MDR is occurring. Future studies are needed to examine if levels of NALP1 vary in GLC4 cells with and without treatment with doxorubicin and in normal lung cells. The cellular location (nucleus or cytoplasm) of the interactions should also be identified. Furthermore, immunoprecipitation of proteins interacting with NALP1 should include VPARP and perhaps identify other proteins interacting in the signaling pathways under MDR and normal culture conditions. This information may contribute insight into the function of VPARP and vaults within the cell. / Department of Biology

Doctor of Philosophy (PhD) / The H69CIS200 cisplatin-resistant and H69OX400 oxaliplatin-resistant cell lines developed as part of this study, are novel models of low-level platinum resistance. These resistant cell lines do not have common mechanisms of platinum resistance such as increased expression of glutathione or decreased platinum accumulation. Rather, these cell lines have alterations in their cell cycle allowing them to proliferate rapidly post drug treatment in a process known as ‘regrowth resistance’. This alteration in cell cycle control has come at the expense of DNA repair capacity. The resistant cell lines show a decrease in nucleotide excision repair and homologous recombination repair, the reverse of what is normally associated with platinum resistance. The alterations in these DNA repair pathways help signal the G1/S checkpoint to allow the cell cycle to progress despite the presence of DNA damage. The decrease in DNA repair capacity has also contributed to the development of chromosomal alterations in the resistant cell lines. Similarities in chromosomal change between the two platinum resistant cell lines have been attributed to inherent vulnerabilities in the parental H69 cells rather than part of the mechanism of resistance. The H69CIS200 and H69OX400 resistant cells are cross-resistant to both cisplatin and oxaliplatin. This demonstrates that oxaliplatin does not have increased activity in low-level cisplatin-resistant cancer. Oxaliplatin resistance also developed more rapidly than cisplatin resistance suggesting that oxaliplatin may be less effective than cisplatin in the treatment of SCLC. The resistant cell lines have also become hypersensitive to taxol but show no alterations in the expression, polymerisation or morphology of tubulin. Rather, the PI3K/Akt/mTOR pathway is involved in both platinum resistance and taxol sensitivity as both are reversed with rapamycin treatment. mTOR is also phosphorylated in the resistant cell lines indicating that platinum resistance is associated with an increase in activity of this pathway. The mechanism of regrowth resistance in the platinum-resistant H69CIS200 and H69OX400 cells is a combination of activation of PI3K/Akt/mTOR signalling and alterations in control of the G1/S cell cycle checkpoint. However, more work remains to determine which factors in these pathways are governing this novel mechanism of platinum resistance.

Aim: The aim of this thesis was to measure a large spectrum of biomarkers in serum or plasma of patients with operable stage of NSCLC and to evaluate and compare the clinical utility of these biomarkers in the three most important clinical applications for NSCLC: diagnosis, prognosis and postsurgery follow up care. Patients and methods: Total of 22 biomarkers with the most promising profiles were monitored: 8 standard tumor markers (cytokeratines Cyfra 21-1, TPA, TPS, and MonoTotal, CEA, SCC, TK, Chromogranin A) and 14 potential useful biomarkers including pro-inflammatory cytokines IL-6, IL-8, MCP-1, pro-angiogenic cytokine VEGF, matrix metaloproteinases MMP-1, MMP-2, MMP-7, MMP-9 and their inhibitors TIMP-1 and TIMP-2, adhesion molecules ICAM-1, VCAM-1, growth factor IGF-1, and PAI-1 stimulating tumor growth and angiogenesis. With a view of evaluating the clinical relevance of these markers for NSCLC we measured serum or plasma levels of these 22 markers in group of 93 patients with NSCLC undergoing radical surgery and in group of 20 patients with benign lung disease. For biomarker measurement were used conventional immunoanalytic routine methods (IRMA, REA, CLIA, MEIA, TRACE, ELISA) and multiplex immunoanalytic method. Results: Cyfra 21-1, MonoTotal, TPA, TPS, CEA, SCC, Chromogranin A, TIMP-1, MMP-1,...

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are highly effective therapies for sub-populations of non-small cell lung cancers. Specific mutations have been identified in the EGFR gene such as L858R which overstimulate cell pathways that lead to tumor growth. All tumors eventually develop resistance to this treatment, rendering them useless, and tumor growth progresses. Escape mutations in the EGFR gene were first seen in patients undergoing treatment with first-generation TKI erlotinib and gefitinib. T790M is a widely seen gate-keeping mutation which overcomes inhibition from erlotinib and gefitinib. Third-generation irreversible TKI, osimertinib, can inhibit tumor cells with this gate-keeping mutation thus overcoming a major hurdle in containment of tumor growth. Unfortunately, patients eventually develop resistance to osimertinib, exhausting options for managing non-small cell lung cancer. Here we analyzed H1975 cells which harbor L858R + T790M mutations. We aimed to track genomic, transcriptomic, and proteomic changes to uncover mechanisms cells use to develop resistance to osimertinib. We established cell colonies which were able to survive high dose treatment up to 2 µM osimertinib. We also saved cells with IC50 of 30 nM to represent drug-tolerant cells. We conducted single-cell sequencing of mRNA transcription and performed hierarchal gene analysis which identified CD74 as a novel factor which was upregulated in drug-tolerant cells. Further we showed CD74 gene was accessible as open chromatin for easy upregulation. Western blot analysis showed increased expression of CD74 after 24 hours of osimertinib treatment. Using siRNA in H1975 cells, we conducted knockdown experiments of CD74 during osimertinib treatment and showed reduced viability. Next, H1975 cells lines were engineered with deletions in CD74 to knockout its expression. These cells also showed reduced viability in the presence of osimertinib. Quantification of apoptosis using caspase-glo assays showed greater activation of apoptosis in cell populations without CD74 compared to normal H1975 cells. H1975-CD74 knockout cells also took longer to become resistant to osimertinib when compared with control. These results show the role of CD74 in helping tumor cells survive EGFR TKI treatment. / 2023-12-05T00:00:00Z

Lung cancer is the leading cause of cancer death worldwide with 2.2 million new cases diagnosed and 1.8 million deaths per year. Lung squamous cell carcinoma (LSCC) is an aggressive histological subtype of non-small cell lung cancers (NSCLC), which is strongly associated with cigarette smoking and exposure to environmental pollutants. In collaboration with the Computational Biomedicine group at Boston University, we identified several putative cancer driver mutations in benign premalignant lung tumors, extracted from upper bronchial airway epithelium. The gene mutations from premalignant tumors are thought to initiate neoplasia but cannot promote malignancy independently. It is hypothesized that additional cooperating mutations will have a compounding effect on tumorigenesis if co-expressed in the same tumor cell. We used cancer genomics data from LSCC primary tumors in the Cancer Genome Atlas (TCGA) database to identify lung pre-malignancy associated genes that are significantly co-mutated. Two of the identified mutant genes, PIK3CA and NFE2L2, were shown to co-occur at a statistically significant rate in LSCC primary tumors. The PIK3CA gene encodes the PI3K lipid kinase, which regulates the AKT and mTOR kinase signaling pathways, thus promoting cell proliferation and survival. NRF2, the product of NFE2L2 gene, is a transcription factor that regulates the antioxidant response, playing a protective role against oxidizing cellular damage. NRF2 promotes the transcription of key proteins in the antioxidant response such as glutathione S transferase and NADPH oxidase. NRF2 is normally subject to ubiquitin-mediated degradation, which is regulated by the KEAP1 protein. Loss of function KEAP1 gene mutations are common in lung cancer. When cells are exposed to oxidizing agents, KEAP1 is modified by these agents, resulting in release and stabilization of NRF2, and the subsequent transcription of antioxidant response genes. Studies of PI3K and NRF2, and their downstream effectors have shown that both the PI3K/AKT/mTOR and NRF2/KEAP1 signaling pathways control autophagy, which is a catabolic process that regulates the recycling of macromolecules under conditions of nutrient deprivation. PI3K and NRF2 both control the activity of the SQSTM1/p62 protein, which plays a major role in autophagic degradation of cargo proteins. Autophagy has been implicated as a tumor suppressive mechanism. Both PI3K and NRF2 are known to inhibit autophagy in lung cancer cells. Based on the significant frequency of co-occurrence of PIK3CA and NFE2L2 gene mutations in pre-malignant LSCC lesions, we hypothesize that PI3K and NRF2 cooperate to inhibit autophagy to promote LSCC progression. To test our hypothesis, we co-expressed mutant forms of PIK3CA (E545K) and NFE2L2 (T80K) into a non-transformed Human Bronchial Epithelial Cell line (HBEC-3KT). We performed a series of Western Blots to verify PI3K and NRF2 protein expression as well as downstream AKT activation and markers of autophagy pathway activation. mTORC1 is an effector of PI3K and plays a central role in the inhibition of autophagy through the PI3K/AKT/mTOR signaling network. Therefore, we performed Western Blot analysis of samples treated with the mTORC1 inhibitor Everolimus to compare the effects of mTORC1 inhibition on autophagy activation in control, single PIK3CA, NFE2L2 and double mutant HBEC3-KT cells. We observed significant suppression of autophagy in the PI3K/NRF2 double mutant cells. Moreover, the studies also showed that the double mutant cells are more sensitive to anti-proliferative effects of Everolimus compared to control and single mutant cells. Taken together, our studies show that PIK3CA and NFE2L2 mutations cooperate to hyperactivate the AKT kinase and to suppress autophagy pathway activation. This represents a key mechanism of the malignant transformation of benign premalignant LSCC lesions. This warrants further research into the cooperation between PI3K and NRF2 in lung cancer pathogenesis. Our results have important implications both for diagnosis and treatment of LSCC. Though many important advances in the treatment of lung cancer have been made over the past few decades including the use of tyrosine kinase inhibitors (TKIs) such as Erlotinib, there is still much to understand about the biology and mechanisms of the disease.1 Blockers of the T-cell checkpoint, such as anti-PD-1 drugs are currently FDA-approved first lines of therapy for NSCLC. In addition, immunotherapy has shown some efficacy in lung cancer patients.2 Our studies provide rationale for the development of therapeutics that suppress NRF2 and PI3K activity in the treatment of LSCC.3 Since mTORC1 inhibitors cause robust inhibition of PIK3CA/NFE2L2 double mutant cell proliferation, future studies will be aimed at testing combinations of mTORC1, PI3K and NRF2 pathway inhibitors to treat LSCC.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19620号 / 医博第4127号 / 新制||医||1015(附属図書館) / 32656 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 森田 智視, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM