The role of NAD(P)H:quinone oxidoreductase 1 in non-small cell lung cancer tumorigenesis and lung cancer stem cell maintenance

Lung cancer is the leading cause of cancer related deaths worldwide. The five-year survival rates for those patients suffering from non-small cell lung cancer (NSCLC), continues to be an abysmal 15%. One of the major reasons for the poor survival rate amongst NSCLC patients is the lack of early detection and subsequent late stage initial diagnosis. Tumors discovered at later stages are often refractory toward chemotherapy and radiation regimens. One theory as to why tumors become resistant to therapy relies heavily on the cells that make up the cancer stem cell (CSC) niche. This small percentage of cells within the heterogeneous tumor has been reported to be responsible for drug resistance, tumor recurrence, and metastasis. In general, CSCs have been isolated using a number of different markers, including cluster differentiation markers, somatic stem cell markers, as well as a number of functional markers such as the side population and aldehyde dehydrogenase (ALDH) activity. While some cancer types, such as breast and hematologic cancers, have been significantly investigated to identify and define their CSC population, lung cancer researchers have only recently begun to identify CSC markers in lung tumors. In addition to the CSC population, malignant cells can also alter their expression of a number of cytoprotective genes that promote tumorigenesis. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a detoxifying enzyme that has been demonstrated to be highly overexpressed in a number of different malignancies. This overexpression has been utilized as a drug target, as the enzyme is expressed at low levels in normal tissue. To this point, there has been success in using NQO1 as a drug target, however little research has been conducted on understanding why NQO1 is overexpressed in these malignancies. The work presented here investigated the role of NQO1 in tumorigenesis as well as its role in maintaining the CSC population in NSCLC. We demonstrate that NQO1 promotes anchorage-independent growth, invasion, reactive oxygen species regulation, anoikis resistance, proliferation, in vivo tumor growth, survival, and ALDH activity. Secondly, we demonstrate that NQO1 also promotes spheroid formation, both in initial and serial contexts, enhances the CSC frequency, and protects spheroid-cultured cells from chemotherapy. Finally, we provide preliminary data that indicates that NQO1 mRNA may be playing an important signaling role in the promotion of the CSC phenotype. This was demonstrated by CRISPR-Cas9 genetic knockout of NQO1 that resulted in a reemergence of the CSC phenotype that can be reversed with transient knockdown of NQO1 mRNA. In summary, our data demonstrate that NQO1 is playing a vital role in the promotion of NSCLC tumorigenesis, as well as supporting the cancer stem cell population. Interestingly, these results may be due to a novel signaling mechanism by NQO1 mRNA, and not the enzyme itself. Further research will be needed to completely understand the role of NQO1 mRNA in NSCLC tumorigenesis and the CSC phenotype

NAD(P)H Quinone Oxidoreductase-1 Expression Promotes Self-Renewal and Therapeutic Resistance in Non-Small Cell Lung Cancer

Identifying cellular drivers responsible for enhancing cancer cell resistance to therapeutics provides critical information for designing more effective drugs. Populations of slowly growing, self-renewing, chemo-resistant cells purportedly contribute to the development of therapeutic resistance in many solid tumors. In the current study, we implemented a tumor spheroid model to determine whether NAD(P)H quinone oxidoreductase-1 (NQO1) was requisite for self-renewal and promotion of the drug-resistant phenotype in non-small cell lung cancer (NSCLC). We found that stable depletion of NQO1 in A549 and H358 human NSCLC tumor models inhibits self-renewal capabilities, as demonstrated by a reduced ability to form primary, secondary, and tertiary spheroids. In contrast, the rescue of NQO1 expression restored the tumor cells’ ability to form spheroids. Additionally, we discovered that NQO1 depletion renders cisplatin-refractory tumor spheroids highly susceptible to drug treatment. Together, these results suggest that NQO1 loss reduces the self-renewing capabilities of NSCLC cells and enhances their susceptibility to clinically relevant therapeutics. These findings describe a novel role for NQO1 and suggest that combining NQO1-inhibitors with conventional chemotherapeutics may enhance anti-tumor effects

Abstract 2503: NQO1's role in maintaining the cancer stem cell phenotype in NSCLC

Abstract NAD(P)H:quinone oxidoreductase 1 (NQO1) is a phase II detoxifying enzyme responsible for quinone reduction, where it scavenges quinone induced reactive oxygen species. In recent studies, NQO1 has been investigated as a possible drug target due to its overexpression in a number of solid tumors. To this end, treatment with the quinone-analog drug, β-lapachone (ARQ-761), has progressed to Phase II clinical trials. In an effort to better understand the necessity of NQO1 in the overall lifecycle of cancer, our earlier work demonstrated that NQO1 played a vital role in a number of tumorigenic processes including anoikis resistance, and most interestingly alteration of aldehyde dehydrogenase (ALDH) activity. Given that ALDH is a widely reported cancer stem cell marker, and loss of NQO1 leads to a decrease in ALDH activity, we have begun to investigate the effect of NQO1 expression on the non-small cell lung cancer (NSCLC) stem cell population. Here, we present data that demonstrates NQO1 is vital to tumorsphere formation as demonstrated by decreased spheroid formation following NQO1 knocked down with shRNA. We also show that NQO1 appears to be necessary for cancer stem cell renewal as illustrated by decreased serial tumorsphere formation. In addition to these aforementioned data, in extreme limited dilution assays we demonstrate a reduced cancer stem cell frequency in NQO1 knockdown cells as compared to controls. Interestingly, in NQO1 knockdown populations, those cells that do form spheres show a remarkable re-expression of NQO1 as well as a rescue of ALDH activity, further supporting NQO1's role in tumorsphere formation and stem cell maintenance. Future work on this project will involve use of the CRISPR-Cas9 system to generate NQO1 knockout cell lines, drug resistance studies, cancer stem cell marker validation, and in vivo limited dilution assays to definitively demonstrate NQO1's necessity in maintaining the NSCLC stem cell population. Citation Format: Brian Madajewski, Erik A. Bey. NQO1's role in maintaining the cancer stem cell phenotype in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2503.</jats:p

The role of NAD(P)H:quinone oxidoreductase 1 in non-small cell lung cancer tumorigenesis and lung cancer stem cell maintenance

Lung cancer is the leading cause of cancer related deaths worldwide. The five-year survival rates for those patients suffering from non-small cell lung cancer (NSCLC), continues to be an abysmal 15%. One of the major reasons for the poor survival rate amongst NSCLC patients is the lack of early detection and subsequent late stage initial diagnosis. Tumors discovered at later stages are often refractory toward chemotherapy and radiation regimens. One theory as to why tumors become resistant to therapy relies heavily on the cells that make up the cancer stem cell (CSC) niche. This small percentage of cells within the heterogeneous tumor has been reported to be responsible for drug resistance, tumor recurrence, and metastasis. In general, CSCs have been isolated using a number of different markers, including cluster differentiation markers, somatic stem cell markers, as well as a number of functional markers such as the side population and aldehyde dehydrogenase (ALDH) activity. While some cancer types, such as breast and hematologic cancers, have been significantly investigated to identify and define their CSC population, lung cancer researchers have only recently begun to identify CSC markers in lung tumors. In addition to the CSC population, malignant cells can also alter their expression of a number of cytoprotective genes that promote tumorigenesis. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a detoxifying enzyme that has been demonstrated to be highly overexpressed in a number of different malignancies. This overexpression has been utilized as a drug target, as the enzyme is expressed at low levels in normal tissue. To this point, there has been success in using NQO1 as a drug target, however little research has been conducted on understanding why NQO1 is overexpressed in these malignancies. The work presented here investigated the role of NQO1 in tumorigenesis as well as its role in maintaining the CSC population in NSCLC. We demonstrate that NQO1 promotes anchorage-independent growth, invasion, reactive oxygen species regulation, anoikis resistance, proliferation, in vivo tumor growth, survival, and ALDH activity. Secondly, we demonstrate that NQO1 also promotes spheroid formation, both in initial and serial contexts, enhances the CSC frequency, and protects spheroid-cultured cells from chemotherapy. Finally, we provide preliminary data that indicates that NQO1 mRNA may be playing an important signaling role in the promotion of the CSC phenotype. This was demonstrated by CRISPR-Cas9 genetic knockout of NQO1 that resulted in a reemergence of the CSC phenotype that can be reversed with transient knockdown of NQO1 mRNA. In summary, our data demonstrate that NQO1 is playing a vital role in the promotion of NSCLC tumorigenesis, as well as supporting the cancer stem cell population. Interestingly, these results may be due to a novel signaling mechanism by NQO1 mRNA, and not the enzyme itself. Further research will be needed to completely understand the role of NQO1 mRNA in NSCLC tumorigenesis and the CSC phenotype.</jats:p

Abstract A64: NQO1 depletion in non-small lung cancer cells decreases their tumorigenicity by reducing the ALDH (high) cancer stem cell population

Abstract Lung cancer is the leading cause of cancer related deaths in the world. In 2015, roughly 160,000 people in the U.S. alone will succumb to the disease. The high mortality is mainly due to late diagnosis and therapeutic resistance, followed by disease progression. One mechanism by which lung cancer is able to circumvent treatment is through the existence of cancer stem cells (CSCs). CSCs are a subpopulation of cells within the heterogenous tumor that are capable of self-renewal, are less sensitive to therapeutics and are believed to be responsible for disease progression, metastasis, and relapse. Aldehyde dehydrogenase (ALDH) high activity has been linked to the cancer stem cell phenotype in various cancers; and the elimination of cells within the heterogenous tumor that have ALDH (high) activity has been associated with decreased tumorigenicity. NADPH quinone oxidoreductase-1 (NQO1) is a two-electron oxidoreductase that is highly overexpressed in many cancers including non-small cell lung cancer. In normal cells NQO1 is expressed only at low levels, but plays a significant role in regulating oxidative stress. Thus, we hypothesized that the noted overexpression of NQO1 in most cancers may be due to its critical role in tumor survival. Specifically, in balancing the noted elevated levels of oxidative stress observed in cancer cells. Thus, reducing NQO1 expression in tumor cells may tip the balance towards pro-death instead of pro-survival. In our studies we used two different shRNA constructs to deplete NQO1 expression and assayed the ability of the knockdown cells to perform in assays that define a tumors ability to participate in disease progression. In brief, our data showed that NQO1 depleted non-small cell lung cancer cell lines had decreased colony formation in soft agar assays, increased detachment induced cell death (anoikis), increased oxidative stress and increased apoptosis. In addition, shNQO1 knockdown caused a depletion in ALDH (high) activity, suggesting that the cancer stem cell population was reduced due to the loss of NQO1 expression. Our in vivo data supported our in vitro data since athymic mice bearing shQNO1 tumors had significantly longer long-term survival and less tumor growth as compared to mice bearing vector-control tumors. In conclusion, our data strongly suggest that NQO1 depletion is a viable target for lung cancer stem cell reduction. Furthermore, our data suggest that implementation of NQO1-directed therapies to reduce cancer stem cells will lead to improved survival in patients whose tumors overexpress NQO1. Citation Format: Brian Madajewski, Michael A. Boatman, Erik A. Bey. NQO1 depletion in non-small lung cancer cells decreases their tumorigenicity by reducing the ALDH (high) cancer stem cell population. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr A64.</jats:p

Depleting Tumor-NQO1 Potentiates Anoikis and Inhibits Growth of NSCLC

The fundamental role that NAD(P)H/quinone oxidoreductase 1 (NQO1) plays, in normal cells, as a cyto-protective enzyme guarding against stress induced by reactive oxygen species (ROS) is well documented. However, what is not known is whether the observed overexpression of NQO1 in neoplastic cells contributes to their survival. The current study discovered that depleting NQO1 expression in A549 and H292 lung adenocarcinoma cells caused an increase in ROS formation, inhibited anchorage-independent growth, increased anoikis sensitization and decreased 3-D tumor-spheroid invasion. These in vivo data further implicate tumor-NQO1 expression in a pro-tumor survival role, since its depletion suppressed cell proliferation and decreased lung tumor xenograft growth. Finally, these data reveal an exploitable link between tumor-NQO1 expression and the survival of lung tumors since NQO1 depletion significantly decreased the percentage of ALDH(high) cancer cells within the tumor population

NAD(P)H Quinone Oxidoreductase-1 Expression Promotes Self-Renewal and Therapeutic Resistance in Non-Small Cell Lung Cancer

Identifying cellular drivers responsible for enhancing cancer cell resistance to therapeutics provides critical information for designing more effective drugs. Populations of slowly growing, self-renewing, chemo-resistant cells purportedly contribute to the development of therapeutic resistance in many solid tumors. In the current study, we implemented a tumor spheroid model to determine whether NAD(P)H quinone oxidoreductase-1 (NQO1) was requisite for self-renewal and promotion of the drug-resistant phenotype in non-small cell lung cancer (NSCLC). We found that stable depletion of NQO1 in A549 and H358 human NSCLC tumor models inhibits self-renewal capabilities, as demonstrated by a reduced ability to form primary, secondary, and tertiary spheroids. In contrast, the rescue of NQO1 expression restored the tumor cells’ ability to form spheroids. Additionally, we discovered that NQO1 depletion renders cisplatin-refractory tumor spheroids highly susceptible to drug treatment. Together, these results suggest that NQO1 loss reduces the self-renewing capabilities of NSCLC cells and enhances their susceptibility to clinically relevant therapeutics. These findings describe a novel role for NQO1 and suggest that combining NQO1-inhibitors with conventional chemotherapeutics may enhance anti-tumor effects.</jats:p

Supplemental Figures 1-14 from Depleting Tumor-NQO1 Potentiates Anoikis and Inhibits Growth of NSCLC

Supplemental Figure 1: Tumor-NQO1 overexpression leads to poor prognosis in lung cancer patients. Supplemental Figure 2: shNQO1-B knockdown of NQO1 leads to decreased growth in soft agar. Supplemental Figure 3: shNQO1-B knockdown of NQO1 in H292 cells leads to decreased growth in soft agar. Supplemental Figure 4: Dicoumarol inhibits growth of A549 cells in soft agar. Supplemental Figure 5: Mac220 inhibits growth of A549 cells in soft agar. Supplemental Figure 6: Stable overexpression of NQO1 in H596 (NQO1 null) lung cancer cells causes increased growth in soft agar. Supplemental Figure 7: Transient knockdown of NQO1 in H596 LPC-NQO1 cells causes decreased growth in soft agar. Supplemental Figure 8: shNQO1-B knockdown of NQO1 in A549 cells decreases cell invasion. Supplemental Figure 9: Stable overexpression of NQO1 in H596 NQO1 null cells causes increased invasion. Supplementary Figure 10: Transient knockdown of NQO1 using siRNA inhibits invasion of HCC1171 cells. Supplemental Figure 11: Stable knockdown of NQO1 in A549 cells causes loss of PARP- 1 protein expression. Supplemental Figure 12: Knockdown of NQO1 reduces ALDH (high) activity in Pancreas cancer cells. Supplemental Figure 13: Knockdown of NQO1 reduces ALDH (high) activity in Prostate cancer cells. Supplemental Figure 14: NQO1 knockdown does not inhibit short-term viability our long-term survival of non-transformed human bronchial epithelial cells.</p

Supplemental Figure Legends from Depleting Tumor-NQO1 Potentiates Anoikis and Inhibits Growth of NSCLC

Supplemental Figure Legends 1-14</p