​

Analysis:

Our clustering and data mining methods provided us with insight into the overlap in e-cigarette vapor and cigarette smoke affected pathways. The majority of the highly significant genes (p<0.001) that were correlated to increasing tumor growth, differed between the two clusters. The overlapping gene that had a high expression index for tumor development was CDKN1A (cyclin-cyclin-dependent kinase). CDKN1A plays a role in the regulation of cell cycle progression and the p53 signaling pathway. Furthermore, we found that specifically the lung cancer cell proliferation pathway was over-indexed tumor growth and was found in both the e-cigarette and cigarette clusters that had a higher representation of tumor growth.  

 

For cigarettes, we focused on cluster 5 which had the highest expression index for tumor growth. Genes PTGS2 and MMP9, as well as the glucose metabolism and inflammatory cytokines pathways had the highest expression in cluster 5 for cigarettes. For e-cigarettes, we repeated this process of finding the cluster with the highest expression index, which was cluster 3. In this cluster, we found that the NFKB1 and JUN genes and the angiogenic growth factors as well as the oncogenes and tumor suppressors pathways, displayed high expression

 

For the laboratory portion of our study, we were able to draw some significant conclusions from the results of our western blot, for all 3 antibodies. We found that proteins, Fibronectin 1 (FN1), GRAM domain protein 4 (GRAMD4), and Phosphoserine Aminotransferase 1 (PSAT1) were all significantly upregulated in smoker cell lines in comparison to nonsmoker cell lines (p-value<0.001).  FN1 is most commonly involved in cell adhesion and migration processes like embryogenesis, wound healing, blood coagulation, host defense, and metastasis. It is involved in both the lung cancer and the oncogenes and tumor suppressor pathways. GRAMD4 is a mitochondrial effector of E2F1-induced apoptosis, or cell-death. This explains the key role of GRAMD4 in the apoptosis pathway. PSAT1 is involved in the glucose metabolism pathway; it affects the proliferation of cells. All 3 proteins have a direct relationship with cancer development and are seen to be upregulated in cigarette smoke caused lung cancer cells. This leads us to the suggestion that cigarette smoke can cause lung tumor development, a theory supported by years of research on cigarettes.

 

Conclusion:

Based on the significant data we studied in this project, we were able to make a series of conclusions about the effects of e-cigarettes on the human body compared to cigarettes and its relation to tumor development. Based on our clustering algorithm, the genes with high tumor growth expression seen in e-cigarette clusters, caused over-indexed tumor growth in the lung cancer cell proliferation pathway. This suggests that e-cigarette vapor may influence the progression of lung cancer, just as cigarettes have, even without tobacco content. For our lab data, we found that, as we predicted, all the proteins (FN1, GRAMD4, and PSAT1) were significantly upregulated in the smoker cell lines, when compared to the nonsmoker cell lines (p<0.001). This signifies that all these proteins can play a role in the development of lung cancer (and other cancers like colon and liver cancer) in smokers when compared to the data of nonsmokers. Since they are all involved in the lung cancer pathway, we can suggest that these proteins will have a similar response in e-cigarette users as well, because based on the cluster analysis, the lung cancer pathway gene expression changes are similar between e-cigarette and cigarette data.

 

In addition, we found that the gene, CDKN1A, was the key overlapping gene between cigarette and e-cigarette clusters.  Both e-cigarettes and cigarettes displayed effects on the p53 signaling pathway as well as the regulation of the cell cycle at the G1 phase, both of which involve the gene CDKN1A. The G1 phase is a part of interphase, where the cell synthesizes mRNA (messenger RNA) and additional proteins, in preparation for mitosis (cell division). CDKN1A inhibits the activity of cyclin-cyclin-dependent kinase 2, therefore regulating the G1 phase. The CDKN1A protein is currently associated with disease like Gastrointestinal Neuroendocrine Tumors and Tongue Cancer, which suggests that e-cigarette and cigarette exposure tends to promote tumor growth, as supported with the high tumor expression indices found in our data.

 

 

We also found that in our cigarette clusters, genes PTGS2 and MMP9, were highly expressed, as well as the glucose metabolism and inflammatory cytokines pathways. Significant research on the glucose metabolism pathway has found that the growth of tumors reprograms this pathway and upregulates it to promote nutrient uptake for the cancer cells; research on inflammatory cytokines also suggests that tumor growth causes inflammation and as a result leads to an increase in inflammatory cytokines. Both these pathways are over-indexed in our cluster study which shows a direct correlation with a higher probability of developing cancer due to change in gene expression in these pathways. The PTGS2 and MMP6 were genes that we also found to exhibit high indexation for the development for tumors, and PTGS2 has a role in the inflammatory pathway and MMP6 has a role in the glucose metabolism pathway, which emphasizes the significance of these pathways tumor development affected by cigarette smoke. Because we found significance in the effects of e-cigarettes on the glucose metabolism pathway, there is room to study whether the use of e-cigarettes increases in the severity of negative impacts on diabetes patients.  

 

Our e-cigarette data suggested the importance of the angiogenic growth factors, oncogenes and tumor suppressors pathways, and NFKB1 and JUN genes in the growth of tumors. Angiogenic growth factors were found to have a high index of expression in the e-cigarette cluster that is seen to promote higher amounts of tumor development; this growth factor pathway is known for leading to blood vessel restriction, blocking of receptors on endothelial cells, and reducing the immune response, all of which promote the growth of tumors in the body. The other pathway, oncogenes and tumor suppressors, was also over-indexed in the expression of tumor-inducing pathways, and this pathway directly relates with the development of tumors as it causes the upregulation and downregulation of genes that regulate the growth of cells. The NFKB1 and JUN genes were found to have a high index in the cluster that shows an increased expression of tumor growth, and since the NFKB1 and JUN genes are in the inflammatory response and tumor pathway and the angiogenic growth factor pathway, respectively, it furthermore supports our results in the relevance of these genes and pathways in the growth of tumors in e-cigarette exposed cells, and how it differs from cigarette exposed cells.

 

FN1 was involved in the oncogenes and tumor suppressors pathway, which can indicate that it may have a strong protein expression rate in e-cigarette users, potentially leading to the development of cancer. The other two proteins, GRAMD4 and PSAT1, are involved in the apoptosis pathway and glucose metabolism pathway, respectively, and since this was not prominently found in e-cigarettes, it is not possible to state with full confidence that the results will be more emphatic in the e-cigarette exposed cells. Overall, due to the common pathway of lung cancer, it can be suggestively inferred that these three proteins will also be upregulated in e-cigarette cells and can potentially lead to tumor development. Although we don’t know which element of e-cigarette toxicity is causing this tumor growth, it is something we can study in the future using these findings.