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,...

Abstract Failure to maintain an appropriate balance between cell death and proliferation is partly due to derangements in the regulation of apoptosis. In this work, apoptosis and the expression of apoptosis regulating proteins were studied by 3' - end labeling of fragmented apoptotic DNA (TUNEL) and immunohistochemistry in a set of 147 tissue samples consisting of 44 biopsies of normal and dysplastic bronchial epithelium, and 103 non-small cell lung carcinomas. The quantity of apoptotic cells and bodies, apoptotic index (AI%), is defined as a percentage of apoptotic cells in the entire tumor cell population. Changes in the apoptotic activity were already seen in the metaplasia-dysplasia-carcinoma sequence of the lung, where the AI% increased gradually until moderate epithelial dysplasia but started to decrease after that. Thus, the AI% for invasive NSCLC (1.20 for squamous cell carcinoma and 1.24 for adenocarcinoma) was slightly lower than in premalignant bronchial epithelium (mean 1.50), but clearly higher than in normal tissue (0.20 for normal bronchial epithelium and 0.24 for lung interstitial cells). 53% of SQCCs and 50% of ACs showed p53 positive nuclei indicative of mutated p53 protein. The immunostaining of bcl-2, bax and mcl-1 revealed diffuse, cytoplasmic staining and was present in most tissues studied. No statistically significant associations between the extent of apoptosis and the expression of p53, bcl-2, bax, or mcl-1 could be found, although . The immunostaining for caspases 3, 6 and 8 was restricted to the tumor areas, reflecting increased apoptotic activity in them. The AI% was significantly higher in NSCLCs in which the single-cell staining pattern for caspase-8 was dominant (P = 0.017), whereas the expression of caspases 3 and 6 had no association with apoptosis. The number of apoptotic cells was significantly higher in NSCLC tumors with a high number of CD3+ and CD8+ T-lymphocytes (P = 0.01) and B-cells (P = 0.05). By multivariate analysis, enhanced apoptosis in NSCLC showed a 1.9-fold risk (95% CI 1.04–3.60; P = 0.04) and p53 positivity a 2.3-fold risk (95% CI 1.30–4.10; P = 0.005) for a shortened survival. Both factors appeared as independent prognostic variables. Apoptosis is clearly enhanced in premalignant and malignant lung tissue in comparison with normal tissue. Furthermore, the expression of the apoptosis-regulating genes is different in tumor tissue from that in normal tissue, and some of the changes in their expression can be seen even in the premalignant lesions of the bronchial epithelium. The expression of caspases seen only in tumor tissue implies the activation of the apoptotic mechanisms and, thus, the lowered treshold of tumor cells to undergo apoptosis. Even in the advanced stages of the disease, the immune defense is effective and the cytotoxic action of activated CD8+ T-cells clearly involves apoptosis. Based on these results it is concluded that alterations in the apoptotic activity and changes in the expression of apoptosis-regulating genes are associated with malignant transformation and growth in lung tissue.

Background: Although palliative chemotherapy is the standard of care for patients diagnosed with stage IV NSCLC, patients with a small metastatic burden, “oligometastatic” disease, may benefit from definitive local therapy. Methods: We identified 186 patients (26% of Stage IV patients) prospectively enrolled in our institutional database from 2002-2012 with oligometastatic disease, which we defined as five or fewer distant metastatic lesions at diagnosis. Univariable and multivariable Cox proportional hazards models were used to identify patient and disease factors associated with improved survival. Using propensity score methods, we investigated the effect of definitive local therapy to the primary site on overall survival. Results: Median age at diagnosis was 61 years, 51% of patients were female, 12% had squamous histology, and 33% had N0-1 disease. On multivariable analysis, ECOG performance status ≥2 (hazard ratio [HR] 2.43), nodal status N2-3 (HR 2.16), squamous pathology, and metastases to multiple organs (HR 2.11) were associated with a greater hazard of death (all p<0.01). Number of metastatic lesions and size of primary were not significantly associated with overall survival. Definitive local therapy to the primary tumor was associated with prolonged survival (HR 0.65, p=0.043). Conclusions: Definitive local therapy to the primary tumor appears to be associated with improved survival in patients with oligometastatic NSCLC. Select patient and tumor characteristics, including good performance status, non-squamous histology, and limited nodal disease, may predict for improved survival in these patients.

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

Bladder cancer is the one of the most common malignancies of the genitourinary system and the overwhelming majority of those cases, approximately 90% in the United States(1), are of the urothelial/transitional cell histologic type. Small cell histologic type of bladder cancer is extremely rare with a mean frequency of 0.7% (1), and due to its rarity, there have not been any large phase III clinical trials in order to establish a definitive treatment regimen. We report here one such case of this rare type of bladder cancer and our approach towards treatment. A 69-year-old man had an incidental finding of microscopic hematuria during routine annual testing performed by his primary care physician. He was referred to a urologist for further evaluation, and in the interim, he began to experience symptoms of nocturia, dysuria and gross hematuria. Cystoscopy revealed a 5 cm sessile mass within the bladder and transurethral resection of the tumor was performed. Histopathological analysis of the tumor revealed muscle invasive poorly differentiated urothelial carcinoma with neuroendocrine features suggestive of small cell carcinoma. Follow-up systemic imaging only revealed multiple lesions in the liver, with the largest solitary liver lesion measuring 4.4 x 3.4 cm and no discrete lung lesions. Patient was started on palliative systemic chemotherapy with carboplatin and etoposide and follow-up imaging demonstrated excellent response after four cycles of treatment; however, follow-up imaging after the completion of 6 cycles of treatment demonstrated disease progression. Patient was referred for consideration of enrollment into any clinical trials; however, unfortunately no trials were found to be available. Patient was subsequently offered systemic treatment with single-agent immunotherapy with pembrolizumab. Due to development of left sided hydronephrosis, nephrostomy tube placement was performed and patient was also started on palliative radiation. Primary small cell carcinoma (SCC) of the bladder is an exceedingly rare malignancy and therefore, data is not readily available in order to guide treatment decisions. The most commonly administered regimen consists of etoposide with a platinum agent, and this regimen is extrapolated from the treatment of SCC of the lung. However, as for patients like ours, who had progression of disease in a short interval and are deemed primary treatment (platinum) refractory, the prognosis certainly becomes far more grim and the treatment choices even more limited. In sharing our treatment approach, we hope to be able to provide insight towards potential future treatment choices for this most-challenging diagnosis, primary small cell carcinoma of the bladder. (1) Blomjous CE, et. al. Small cell carcinoma of the urinary bladder. A clinicopathologic, morphometric, immunohistochemical, and ultrastructural study of 18 cases. Cancer. 1989 Sep 15; 64(6):1347-57.