Emerging Role of Nanog in Tumorigenesis and Cancer Stem Cells

Abstract

Nanog is a transcription factor that is well-established as a key regulator of embryonic stem cell (ESC) maintenance. Recent evidence demonstrates that Nanog is dysregulated and intimately involved in promoting tumorigenesis in part through regulation of the cancer stem cell (CSC) population. Elevated Nanog is associated with poorer outcome in numerous epithelial malignancies. Nanog is enriched in CSCs and ablation of Nanog is sufficient to reduce the CSC pool. Nanog has also been implicated to promote chemoresistance and epithelial-mesenchymal transition (EMT). Insight into the Nanog signaling cascade, upstream regulators and downstream effectors, is beginning to emerge but remains to be fully elucidated. This review highlights the current literature on the emerging role of Nanog in tumorigenesis and CSCs.

Introduction

The human Nanog gene (referred to as Nanog1) is located in chromosome 12; however, there are 10 Nanog pseudogenes in the human genome.¹ NanogP8, located in chromosome 15, is the only pseudogene with an intact open reading frame. NanogP8 codes for a protein that is identical to Nanog except for a change in amino acid 253 from glutamine to histidine.² For clarity, in this mini-review, Nanog is a term used without specificity for Nanog1 or its pseudogenes, including NanogP8. We used the terms Nanog1 and NanogP8 to discuss the small number of studies that assessed Nanog1 and/or NanogP8 mRNA expression or promoter activity. At this time, Nanog1 and NanogP8 protein cannot be detected independently since current anti-Nanog antibodies are unable to discriminate between these two proteins due to the high degree of amino acid similarity. A preponderance of studies discussed in this mini-review measured Nanog mRNA expression with PCR primers that amplifies both Nanog1 and NanogP8. Only a limited number of reports used approaches to independently measure Nanog1 and NanogP8 mRNA expression in established cell lines and primary tumor samples. NanogP8 was reported to be primarily responsible for increased Nanog mRNA expression in a diverse panel of carcinoma cell lines.^3,4 In contrast, other studies have shown that Nanog1 and NanogP8 mRNA expression are differentially expressed, albeit at different extent, in colorectal and hepatocellular primary tumors and carcinoma cell lines suggesting that the role of Nanog1 and NanogP8 may be highly tissue-type dependent ^5,6,7. Adding another level of complexity is that hyperactivation of the Nanog1 promoter was sufficient to identify cancer stem cells (CSCs) in a hepatocellular carcinoma cell line⁸. These reports indicate that Nanog1 mRNA expression is not exclusively limited in ESCs but also present in carcinoma cells, including the CSCs. Additional research is needed to better define the global contributions and requirements of Nanog1 and NanogP8 in tumorigenesis and CSCs.

The seminal work implicating Nanog as a potential oncogene was performed in NIH3T3 fibroblasts. Ectopic overexpression of Nanog enhanced NIH3T3 cell proliferation by promoting S-phase entry.⁹ Subsequently, an independent group confirmed that Nanog-overexpressing NIH3T3 cells have increased proliferative capacity and moreover, showed that Nanog is sufficient to promote anchorage-independent growth using the soft agar colony formation assay.¹⁰ Global cDNA microarray analyses revealed that Nanog induces the expression of a sub-set of direct embryonic stem cells (ESC) Nanog-regulated genes in NIH3T3 cells including two well-recognized oncogenes, Signal transducer and activator of transcription 3 (Stat3) and Jun-B.¹⁰ These observations suggest that there is overlap in the Nanog signaling network between ESCs and transformed cells; however, the extent of this overlap remains unclear in carcinoma cells.

Evidence has accumulated to implicate Nanog as a critical player in tumorigenesis. Loss-of-function studies demonstrated that Nanog is essential for numerous epithelial malignancies in part through regulation of the cancer stem cell (CSC) population. The cancer stem cell (CSC) hypothesis posits that CSCs are the unique cells with the potential to expand the CSC pool and differentiate into heterogeneous nontumorigenic cells that constitutes the bulk of the tumor.¹¹ Extensive work has demonstrated that CSCs from primary tumors or established carcinoma cell lines can be isolated from a heterogeneous population using cell surface markers, such as CD44 and CD133, aldehyde dehydrogenase (ALDH) activity using the ALDEFLUOR assay, and Hoechst dyes to identify the side population. Another method to enrich for the CSC population is to form tumorspheres in well-defined, non-adherent cell culture conditions. Our group and others have shown that tumorspheres have higher in vivo tumor incidence rate than parental adherent cells and thus, indicates that CSCs are present at a higher proportion in tumorspheres.¹² There is experimental data that CSCs are resistant to conventional chemotherapy and radiation and may be the cells responsible for disease recurrence and/or progression.¹¹ Thus, further understanding of the role of Nanog in controlling CSCs is warranted and may lead to the development of novel therapeutics to preferentially ablate the CSC population.

Breast and Ovarian Carcinomas

There is accumulating evidence that Nanog plays a critical role in resistance to standard therapy in breast and ovarian carcinoma. The hyaluronan (HA)-CD44 signaling pathway promotes the association between Nanog and Stat3 resulting in activation of a Stat3-driven chemoresistance program through an increase in multiple drug resistance protein 1 (MDR-1) in MCF7 breast carcinoma cells and SKOV3 ovarian carcinoma cells.¹³ Subsequent work from the same group showed that HA activation of CD44 induces protein kinase Cε (PKCε)-mediated phosphorylation of Nanog leading to an increase in miR-21 and a decrease in program cell death 4 (PCD4) in MCF7 cells.¹³ In ovarian carcinoma, Nanog expression was elevated in paclitaxel-resistant compared to parental SKOV3 cells.¹⁴ A recent study demonstrated that miR-214 modulates chemoresistance and CSC population through regulation of the p53/Nanog signaling axis in ovarian carcinoma cells. Nanog expression was shown to be controlled by miR-214 in wildtype p53 OV433 and OV2008 ovarian carcinoma cells but not in p53 mutant OV3 and SKOV3 cells.¹⁵ Ovarian CSCs isolated from primary tumors have elevated Nanog, Oct3/4, and Bmi1 expression and showed enhanced resistance to standard chemotherapeutics, cisplatin and paclitaxel.¹⁶ Similarly, ovarian CSCs isolated using Hoechst 33342 are enriched for Nanog and Oct3/4 expression and are refractory to cisplatin in vitro and in vivo.¹⁷ The reprogramming of non-breast CSCs to breast CSCs by radiation is associated with an increase in the expression of ESC transcription factors, Nanog, Oc3/4, and Klf4.¹⁸ These findings show that Nanog is associated with a chemoresistant phenotype and perhaps, also modulates sensitivity to radiation.

An intriguing report provided insight in the function of Nanog in a transgenic mouse model of breast carcinoma. Mouse mammary tumor virus (MMTV)-Cre/LoxP-mediated overexpression of Nanog in the mammary gland was insufficient to promote tumorigenesis.¹⁹ However, Nanog enhanced mammary epithelial tumor cell proliferation and incidence of distant metastasis in MMTV-Wnt-1 transgenic mice.¹⁹ The observation of increased tumor cell proliferation is consistent with the study demonstrating that Nanog occupies the promoter of cyclinD1 to regulate cell cycle progression and cell proliferation in MCF7 and MDA-MD231 breast carcinoma cells.²⁰ Global cDNA microarray analyses of mammary tumors from Wnt-1 transgenics and double Wnt-1/Nanog transgenics revealed that 116 genes, including genes critical for epithelial-mesenchymal transition (EMT) such as platelet derived growth factor receptor-α, were differentially regulated.¹⁹ Another group showed that Nanog-mediated cell migration and cell invasion is dependent on the regulation of E-cadherin and forkhead box J1 (FOXJ1). FOXJ1 was reported to have tumor-suppressive actions in breast carcinoma;²¹ however, its function in ovarian carcinoma remains to be elucidated. These results provide initial evidence that Nanog promotes EMT, in part, through regulation of PDGFRα and E-cadherin to drive a metastatic tumor cell phenotype.

Several groups studied the utility of Nanog as a prognostic and predictive biomarker (Table 1). Elevated Nanog levels were associated with poor prognosis in breast carcinoma patients.²² In addition, Nanog mRNA expression was reported to be higher in breast carcinoma patients and associated with tumor size, clinical stage, and lymph node status.²⁰ Increased nuclear Nanog levels was associated with chemoresistance and poorer disease-free and overall survival in a cohort of ovarian carcinoma patients treated with adjuvant chemotherapy following surgery.²³ Similarly, elevated Nanog levels were reported to be an independent prognostic biomarker in stage I/II ovarian serous carcinoma patients; median survival of 40 months for high Nanog and 120 months for low Nanog patients.¹⁴

Table 1.

Nanog as a Biomarker in Epithelial Carcinomas

Carcinoma	Study (Reference #)	# of Patients	Parameters/Outcomes
Breast	20	116	Tumor size, p=0.003 Clinical stage, p=0.037
	22	100	DFS, p=0.004 OS, p=0.033
Colorectal	24	175	LN+, p=0.024 OS, p=0.000
Gastric	29	105	LN+, p=0.004 Tumor invasion, p=0.001 OS, p=0.000
Head and Neck	38	52	Grade, p<0.05 OS, p<0.01
	43	46	OS, p<0.0001 (high Nanog/GRP78 vs. low Nanog/GRP78)
	47	78	DFS, p=0.04 (high CTGF, Nanog, Oct3/4, and Sox2)
Lung	56	118	OS, p<0.01
	59	309	Grade, p<0.001 OS, p<0.001
Ovarian	14	74	OS, p=0.02
	23	90	DFS, p=0.002 OS, p=0.001

Colorectal Carcinoma

In a large cohort of colorectal carcinoma patients (n=175), high Nanog protein was associated with positive lymph nodes and Dukes classification.²⁴ Multivariate analyses revealed that increased Nanog levels are an independent biomarker for poor prognosis.²⁴ Consistent with this report using patient specimens, several independent studies support the notion that Nanog is a putative oncogene and elevated in CSCs in colorectal carcinoma. Tumorspheres cultivated from a panel of colorectal carcinoma cells, including CX-1, had an 8- to 122-fold increase in Nanog expression.²⁵ Knockdown of Nanog in CX-1 cells decreased the number of tumorspheres and importantly, hampered in vivo tumorigenicity and metastasis in NOD/SCID mice.²⁵ Consistent with these observations, genetic ablation of Nanog in SW620 colorectal carcinoma cells suppressed cell proliferation in vitro and tumor growth in athymic nude mice.⁵ Conversely, enforced overexpression of Nanog promoted cell invasion and cell migration in SW480 colorectal carcinoma cells.²⁴ Similarly, overexpression of Nanog in HCT116 colorectal carcinoma cells enhanced colony formation and in vivo tumorigenicity.⁶ These reports demonstrate that Nanog play a critical role in colorectal tumorigenesis possibly through control of the CSC population.

Work on colorectal carcinoma provided molecular insight on the components of the Nanog circuitry. Nanog induces the expression of Slug and Snail, two hallmark EMT genes.²⁴ Interestingly, Snail was shown to modulate Nanog expression demonstrating that a positive feedback regulatory mechanism exists between these two proteins.²⁴ c-Jun binds to the Nanog promoter at the octamer M1 response element and cooperate with β-catenin/T-cell factor (TCF) to drive Nanog expression in colorectal carcinoma cells.⁶ In colorectal CSCs, epithelial cell adhesion molecule (EpCAM), a transmembrane protein involved in EMT, and the core ESC transcription factors, Nanog, Sox2, Oct3/4 were elevated in CSCs.²⁶ Knockdown of EpCAM reduced the expression of the core ESC transcription factors and compromised the in vivo tumor initiating rate.²⁶ Lastly, shRNA-mediated ablation of Nanog decreased the expression of Sox2 and Oct3/4 suggesting that Nanog may be the signaling hub that controls the other core ESC transcription factors.²⁵

An intriguing study showed that Nanog may allow a select population of carcinoma cells to evade immune surveillance. An in vitro cytotoxic T lymphocyte (CTL)-mediated selection process resulted in the enrichment of carcinoma cells with CSC-like properties through the Nanog/Tcl1/Akt signaling axis.²⁶ Inhibition of Nanog in HCT116 colorectal carcinoma cells was sufficient to dramatically enhance immune clearance resulting in long-term control of the primary tumor.²⁷ These results provide initial evidence that the role of Nanog in tumorigenesis is multilayered and may involve the escape of CSCs from immune detection and clearance.

Gastric Carcinoma

The first study linking Nanog to gastric carcinoma reported that Nanog1 and its pseudogene, NanogP8, were overexpressed in primary gastric tumors compared to adjacent normal epithelium.²⁸ Subsequently, in a larger cohort (n=105) of surgically-treated gastric carcinoma patients, elevated Nanog levels were associated with positive lymph node status, advance clinical stage, and differentiation.²⁹ Furthermore, gastric carcinoma patients with high Nanog levels had inferior 5-year overall survival.²⁹ These observations demonstrate that Nanog may be a key driver for the development and/or progression of gastric carcinomas.

Several groups demonstrated that Nanog is enriched in gastric CSCs. ALDH and CD44 are two markers reported to identify CSCs in gastric carcinoma cell lines.^30,31 ALDH^high NCI-N87 and SNU-1 gastric carcinoma cells showed CSC features, including enhanced tumorsphere formation, enrichment in the expression of the core ESC transcription factors, Nanog and Sox2, and resistance to cisplatin and 5-flurouracil (5-FU).³² Gastric CSCs identified using the CD44 surface marker in MKN-45 gastric carcinoma cells had elevated levels of Nanog, Sox2, Oct3/4, and Lin28.³³ In addition, a panel of miRNAs was differentially expressed between CD44+ and CD44− MKN-45 cells, including miR-21 and let-7a.³³ Tumorsphere-forming cells derived from MKN-45 are resistant to cisplatin and 5-FU and have increased expression of Nanog, Sox2, Oct3/4, and CD44.³⁴ Importantly, tumorsphere-forming cells were shown to have a 100-fold increase in in vivo tumor initiating potential in athymic nude mice compared to parental MKN-45 cells.³¹ Although these studies indicate that Nanog is associated with a chemoresistant CSC phenotype, it remains to be defined whether Nanog is indispensable for CSC identity and maintenance in gastric carcinomas.

Head and Neck Squamous Cell Carcinoma

Similar to the work in breast carcinoma, hyaluronan (HA) activation of CD44 increased Nanog-Stat3 heterodimerization and occupancy of miR-21 promoter in HSC-3 head and neck squamous cell carcinoma (HNSCC) cells.³⁵ In addition, miR-21 was shown to regulate PCD4 and inhibitor of apoptotic protein (IAP), genes in the apoptotic pathway, to control chemoresistance in HNSCC cells. siRNA-mediated ablation of Nanog or Stat3 effectively blocked HA-induced miR-21 production providing evidence that Nanog and Stat3 are downstream of the CD44 signal transduction pathway. A subsequent study from the same research group showed that treatment of HSC-3 cells with HA enhanced the interaction between CD44v3 with the core ESC transcription factors, Nanog, Oct3/4, and Sox2, leading to miR-302 expression.³⁶ The core ESC transcription factors occupy the promoter region of miR-302 to enhance miR-302 expression.³⁶ miR-302 promoted chemoresistance through control of several inhibitors of apoptosis (IAPs), including cIAP-1, cIAP-2, and XIAP.³⁶ This result is consistent with a study showing that high Nanog and Oct3/4 levels were associated with cisplatin-resistance in a cisplatin-treated HNSCC cohort (n=20).³⁷ Moreover, in an independent study elevated Nanog or Oct3/4 protein was associated with inferior overall survival in a surgical cohort (n=52) of HNSCC patients suggesting that Nanog and Oct3/4 may promote an aggressive phenotype independent from cisplatin resistance.³⁸

miRs play an integral role in Nanog circuitry in HNSCC. miRs, such as miR-21 and miR-302, are regulated by Nanog, whereas, other miRs, such as, let-7a and miR-107, regulate Nanog. let-7a expression was decreased, in contrast, Nanog and Oct3/4 expression were increased in HNSCC tumors compared to adjacent normal epithelium.³⁹ ALDH^high CSCs isolated from primary HNSCC tumors had reduced let-7a and elevated Nanog and Oct3/4 expression. Overexpression of let-7a in ALDH+ HNSCC CSCs inhibited Nanog and Oct3/4 expression, enhanced cis-platinum response, and compromised in vivo tumorigenicity. In the same model system, shRNA-mediated ablation of Nanog suppressed in vivo tumor incidence rate to a similar extent as let-7a overexpression suggesting that Nanog may be the key let-7a downstream gene to modulate the CSC population. miR-107 is a putative tumor-suppressor gene and directly regulates PKCε in HNSCC cells.^40,41 Interestingly, delivery of miR-107 in CAL27 and UMSCC74A HNSCC cells decreased PKCε as expected but also reduced the expression of the core ESC transcription factors.⁴⁰ Overexpression of Nanog was sufficient to rescue the Oct3/4 and Sox2 expression and tumorsphere formation defect in UMSCC74A HNSCC cells treated with exogenous miR-107.⁴⁰ This finding supports the work in colorectal carcinoma showing that Nanog is upstream of Oct3/4 and Sox2 to further confirm the hierarchy of the core ESC transcription factor in carcinoma cells.²⁵ It is clear that let-7a and miR-107 regulate the core ESC transcription factors; however, whether the regulation is through a direct or indirect mechanism remains to be determined.

PKCε was reported to mediate serine phosphorylation of Nanog downstream of an activated HA-CD44 signal to promote a chemoresistant phenotype in MCF7 breast carcinoma cells.⁴² Phosphopeptide mapping demonstrated that PKCε directly phosphorylates Nanog at multiple residues, including T78, S79, S135, T200 and T280.¹¹ Phosphorylation of Nanog at T200 and T280 was shown to modulate Nanog function in UMSCC74A HNSCC cells at multiple levels; protein stability, homodimerization, target gene promoter occupancy, and recruitment of the p300 co-activatior.¹¹ Overexpression of phosphorylation-insensitive T200A or T280A imparted a dominant-negative function in UMSCC74A cells to reduce in vivo tumorigenicity, in part through depletion of the CSC pool.¹¹ These findings provide convincing evidence that post-translational phosphorylation is essential to modulate Nanog activity in HNSCC cells.

Glucose-regulated protein 78 (GRP78), a stress-inducible gene that controls unfolded protein response was reported to be a putative HNSCC CSC marker. Transcriptome analysis revealed that GRP78 is elevated in tumorspheres generated from SAS HNSCC cells suggesting that GRP78 may be utilized as a cell surface marker to identify HNSCC CSCs.⁴³ GRP78+ SAS cells had increased tumorsphere formation potential and are enriched for Nanog and Oct3/4 expression compared to GRP78− SAS cells. In addition, GRP78+ SAS cells demonstrated higher in vivo tumorigenicity; 1 × 10² GRP78+ cells was able to generate a tumor, whereas, tumors did not develop with 1 × 10⁴ GRP78− cells in athymic nude mice. Genetic ablation of GRP78 in SAS cells resulted in a decrease in Nanog and Oct3/4 expression and a reduction in the CSC population. Moreover, high GRP78 levels were associated high Nanog levels in a cohort of surgically-treated HNSCC patient (n=46). HNSCC patients with high GRP78/high Nanog had the worst overall survival.⁴³ GRP78 was reported to inactivate p53 in HNSCC cells and p53 negatively regulates Nanog to induce the differentiation of ESCs.^44,45 These observations suggest that GRP78 may modulate p53 to control Nanog levels in HNSCC.

HNSCC CSCs are enriched for Nanog expression and associated with an EMT phenotype. Tumorspheres generated from a panel of HNSCC cell lines, UD-SCC1, UMSCC11B, UT-SCC9, UT-SCC22, and UT-SCC24A, had a higher percentage of ALDH^high cells and increased expression of the core ESC transcription factors, Nanog, Sox2, and Oct3/4 than parental adherent cells.⁴⁶ Moreover, these HNSCC tumorspheres displayed the hallmark EMT features; high vimentin/low E-cadherin expression signature and enhance cell invasion. Interestingly, Nanog appears to play a role in mesenchymal-epithelial transition (MET), the reverse process of EMT, in HNSCC as well. A recent study showed that connective tissue growth factor (CTGF) promotes MET and reduce invasion in HNSCC cells.⁴⁷ CTGF enhanced the expression of the core ESC transcription factors through c-Jun.⁴⁷ An association between CTGF levels and Nanog/Oct3/4/Sox2 levels was demonstrated in primary tumors from HNSCC patients. Multimarker analyses revealed that co-expression of CTGF, Nanog, Oct3/4, and Sox2 was associated with the worst overall survival. These results suggest that CSCs are not exclusive to EMT cells but may also be present in MET cells. In support of this notion, HNSCC CSCs were reported to exist in two distinct phenotypes. CD44+/ESA+ HNSCC CSCs were proliferative and have epithelial characteristics, whereas, CD44+/ESA− HNSCC CSCs were motile and have EMT features.⁴⁸ Interestingly, non-EMT CSCs and EMT CSCs could switch phenotypes suggesting that the behavior of CSCs is dynamic and not static. Inhibition of glycogen synthase kinase 3β regulates Nanog and promotes the differentiation of EMT CSCs to non-MET CSCs.⁴⁹ Taken together, there is evidence that modulation of Nanog may be a key step in the dynamic regulation of EMT CSCs and non-EMT CSCs.

Hepatocellular Carcinoma

Several studies showed that Nanog expression and levels are increased in hepatocellular carcinoma (HCC) cell lines and primary tumors.^7,50 Elevated Nanog expression was associated with advance disease (TNM Stage III/IV) in a small cohort (n=15) of HCC patients.⁵⁰ Two independent studies demonstrated that Nanog was a prognostic biomarker for unfavorable survival in HCC. In a cohort of 59 HCC patients, high Nanog protein was associated with poorer overall (p=0.044) and disease-free survival (p=0.042).⁸ In another study, patients with high Nanog levels had a median survival of 26.2 months, whereas, patients with low Nanog levels had a median survival of 36.5 months (n=52, p=0.03). The mechanism for Nanog dysregulation in HCC remains to be fully elucidated; however, a recent study provides initial insight that Nanog may be epigenetically regulated at the Nanog1 promoter. In comparison to normal liver cells, Nanog1 promoter was hypomethylated in hepatocellular carcinoma cell lines, PLC, 97L, and 97H.⁵⁰ In line with these cell line results, Nanog1 promoter methylation was reduced in the primary HCC tumors compared to normal adjacent tissue.⁵⁰ In murine ESCs, Nanog promoter was shown to be hypomethylated relative to differentiated cells providing further evidence that epigenetic regulation is utilized to dynamically control Nanog expression and pluripotency.⁵¹ Further research is needed to determine if loss of epigenetic repression of the Nanog1 promoter is a general mechanism used by carcinoma cells and CSCs to enhance Nanog expression.

Similar to the results in HNSCC, Nanog was reported to promote EMT and cell invasion in HCC. Overexpression of Nanog in low Nanog-expressing Huh7 HCC cells resulted in an increase in cell invasion and induced EMT with the hallmark high Vimentin/low E-Cadherin signature.⁷ Furthermore, in vivo tumor growth and intrahepatic metastasis were augmented in Nanog overexpressing-Huh7 cells compared to Huh7 control cells.⁷ Nanog was shown to modulate Nodal, an EMT regulator, expression through direct promoter occupancy. Forward and reverse genetic experiments revealed that Nanog activates the Nodal/Smad3 signaling module to drive EMT and cell invasion in HCC.⁷

There is limited but exciting data to support the notion that Nanog1 promoter hyperactivation can be used as tool to mark CSCs in HCC. Using Nanog1 promoter-GFP as the reporter, Nanog+ cells isolated from Huh7 HCC cells using FACS had higher Nanog levels, as expected and lower levels of mature hepatocyte markers, albumin and glucose-6-phosphatase.⁸ Nanog1+ Huh7 cells possess CSC traits, such as an increase in chemoresistance, self-renewal, tumorsphere formation, and in vivo tumor incidence compared to Nanog1− Huh 7 cells.⁸ Insulin-like growth factor 2 (IGF2) and insulin growth factor receptor 1 (IGF1R) was modulated in Nanog1+ Huh 7 cells suggesting that the insulin growth factor (IGF) pathway may be downstream of Nanog.⁸ Interestingly, IGF1R inhibitors, picropodophyllin and AEW541, decreased the self-renewal and Nanog expression in Nanog1+ Huh7 HCC cells. These results suggest a cross-talk mechanism exists between Nanog and IGF1R to maintain the CSC population in HCC.

Lung Carcinoma

An association between Nanog and lung CSCs was reported by several research groups. CSC-like cells were successfully isolated from a panel of 15 primary lung carcinoma cell lines using CD44.⁵² CD44^high lung carcinoma cells had increased Nanog and Oct3/4 expression and were less sensitive to radiation.⁵² In addition, tumorsphere formation potential was limited to the CD44^high population in these primary lung carcinoma cell lines.⁵² Consistent with the results using primary lung carcinoma cells, Nanog and Oct3/4 levels were enriched in CD44^high compared to CD44^low HT1299 lung carcinoma cells.⁵³ Moreover, 1 × 10⁴ CD44^high H1299 cells were able to generate a tumor in athymic nude mice, in contrast, tumors did not develop with 2 × 10⁵ CD44^low HT1299 cells.⁵³ CD133+ CSCs derived from the primary tumors of non-small cell lung carcinoma patients were reported to have higher Nanog, Sox2, and Oct3/4 expression and phosphorylated Stat3 levels.⁵⁴ Another study showed that enforced overexpression of Nanog and Oct3/4 in A549 cells increased the CD133+ pool, tumorsphere formation, and in vivo tumor incidence.⁵⁵ Interestingly, treatment with cucurbitacin I, a Stat3 inhibitor, altered the stemness gene expression profile of CD133+ CSCs to a differentiated CD133− gene signature.⁵⁴ Stat3 is known to modulate the core ESC transcription factors in ESCs.⁵⁶ Results with cucurbitacin I suggests that Stat3 may control a global set of stemness genes in CSCs as well.

There is evidence that transforming growth factorβ-1 (TGFβ-1) and epidermal growth factor (EGF) regulate Nanog and promote EMT in lung carcinoma cells. TGFβ-1 treatment of LC31 non-small cell lung carcinoma cells induced EMT with the hallmark high Vimentin/low E-cadherin expression profile.⁵⁷ The expression of the core ESC transcription factors, Nanog, Sox2, and Oct3/4, were increased following TGFβ-1 exposure.⁵⁷ Furthermore, TGFβ-1-treated LC31 cells had a dramatic increase in tumorsphere formation efficiency and size and an increase in tumor incidence and growth in NOD/SCID mice.⁵⁷ Treatment of A549 and H23 lung carcinoma cells with EGF induced EMT and resulted in an increase in nuclear levels of Nanog and β-catenin.⁵⁸ In support of this in vitro finding, nuclear Nanog levels were shown to be associated with nuclear β-catenin levels in primary tumors from a large cohort of surgically-treated non-small cell lung carcinoma patients (n=309).⁵⁸ Multivariate analyses revealed that elevated β-catenin and Nanog were independent prognostic biomarkers for overall survival in this patient population. The link between Nanog and EMT was further supported by the study showing that co-overexpression of Nanog and Oct3/4 increased Slug and promoted EMT in A549 cells.⁵⁵ Multimarker analyses revealed that triple Nanog/Oct3/4/Slug-positive patients had the worst outcome, whereas, triple negative cases had the most favorable outcome in a population of lung adenocarcinoma patients.⁵⁵

Prostate Carcinoma

Nanog expression was reported to be elevated in benign prostatic hyperplasia (BPH) and prostate carcinoma specimens compared to normal prostate epithelium.⁵⁹ Interestingly, Nanog expression was similar between BPH and prostate carcinoma tissues suggesting that dysregulation of Nanog may be an early event in the prostate tumorigenesis cascade. Nanog was shown to be predominantly expressed from the NanogP8 pseudogene in a panel of prostate carcinoma cell lines, DU145, LNCaP, and PC3, and primary prostate carcinoma cells.³ NanogP8 expression was enriched by 5-fold in CD133+ and CD133+/CD44+ CSCs compared to non-CSC.³ In addition, NanogP8+ DU145 and PC3 cells identified using a NanogP8 promoter-GFP sensor generated larger tumors than NanogP8− cells.⁶⁰ Genetic ablation of Nanog in DU145 and primary prostate carcinoma cells resulted in a reduction in in vivo tumor incidence and growth.³ Conversely, overexpression of Nanog in LNCaP cells generated larger tumors than control LNCaP cells in castrated NOD/SCID mice suggesting that Nanog may promote an androgen-independent prostate carcinoma phenotype.⁶⁰

The platelet derived growth factor-D (PDGR-D)/Lin28B/Nanog signaling axis was reported to modulate EMT and CSC-like cells in prostate carcinoma cells. Ectopic overexpression of PDGR-D in PC3 cells promoted EMT and increased tumorsphere formation efficiency.⁶¹ Expression profiling revealed that PC3/PDGF-D cells had a global change in EMT-associated genes, including E-cadherin and Vimentin, and ESC-related genes, Nanog, Oct3/4, Sox2, and Lin28B. shRNA-mediated knockdown of Nanog was sufficient to suppress tumorsphere formation in PC3/PDGF-D cells. Moreover, ablation and rescue experiments indicated that Lin28B regulate Nanog through let-7.⁶¹

Telomerase immortalization of human primary prostate tumor cells (HPET) resulted in the expansion of cells without androgen receptor (AR) and p63 expression akin to the prostate stem/progenitor population expression profile.⁶² The core ESC transcription factors, Nanog, Oct3/4, and Sox2, and CSC markers, CD44 and CD133, were expressed in HPET cells. Importantly, HPET cells implanted in NOD/SCID mice reconstituted the heterogeneity of the original patient tumor providing another line of evidence that the HPET cells contain a population of CSCs.⁶² Other studies showed that CD44 and CD133 were associated with high Nanog in prostate carcinoma cell lines. CD44+/CD133+ PC3 cells displayed CSC-like features, such as enhanced tumorsphere formation and elevated Nanog levels.⁶³ Similarly, CD117+/ATP-binding cassette sub-family G member 2 (ABCG2)+ cells isolated from the 22RV1 prostate carcinoma cell line overexpress the core ESC transcription factors, Nanog, Oct3/4, and Sox2, and the CSC marker CD133. This CD117+/ABCG2+ subpopulation was refractory to a panel of standard chemotherapeutics including cisplatin and highly tumorigenic in vivo.⁶⁴

Tumors contain distinct areas that are poorly vascularized or hypoxic. Emerging evidence indicates that CSCs are localized to hypoxic niches suggesting that hypoxia may contribute to CSC identity and maintenance.⁶⁵ DU145 and PC3 cells cultured under hypoxic conditions enhanced hypoxia-inducible factor-1α (HIF-1α) and hypoxia-inducible factor-2α (HIF-2α) as expected but also, increased Nanog expression, predominantly the NanogP8 pseudogene.⁶⁶ HIF-2α was reported to directly regulate Oct3/4 expression through promoter occupancy.⁶⁷ Hypoxia enhanced Nanog expression and levels in prostate carcinoma cells;⁶⁶ however, the molecular mechanism of this observation remains to be determined. HIF-1α was reported to co-localize with Nanog and Oct3/4 in primary tumors from prostate carcinoma patients further supporting a connection between these proteins.⁶⁸ Hypoxia promoted the expansion of the CD44+ CSCs in DU145 and PC3.⁶⁶ It is unclear, at this time, if hypoxia-mediated CSC expansion is through an increase in symmetric CSC division or reprogramming of differentiated tumor cells to CSCs.

Conclusions

Current literature from numerous epithelial malignancies indicates that Nanog has a pleiotropic role in the tumorigenesis cascade, including resistance to chemotherapy and radiation, promotion of EMT, and modulation of the CSC population. Ablation or inactivation of Nanog suppressed the tumorigenic potential of carcinoma cells in vitro and in vivo. In contrast, enforced overexpression of Nanog in established carcinoma cell lines enhanced in vitro and in vivo tumorigenicity. However, it remains to be determined if overexpression of Nanog will be sufficient to transform non-tumorigenic epithelial cells. Wildtype Nanog overexpression was limited to induce a hyper-proliferative epithelial cell phenotype in the mammary glands of MMTV-Nanog transgenic mice.¹⁹ At this time, it is unclear if Nanog is promoting proliferation of differentiated and/or stem/progenitor mammary epithelial cells. Since PKCε directly phosphorylates and activates Nanog, it will be interesting to determine if the double PKCε/Nanog transgenic will have a higher tumor penetrance and/or distant metastasis rate than the respective single PKCε or Nanog transgenics. Alternatively, overexpression of phosphorylation-mimetic Nanog mutants, such as T200D and/or T280D, may be sufficient to promote tumorigenesis and metastasis in a transgenic mouse model. These transgenic mouse models may be crucial to define the role of Nanog in regulation of normal and CSC identity and maintenance.

Nanog circuitry has been an intense area of interest in ESCs and is beginning to be elucidated for carcinoma cells. Multiple oncogenic transduction pathways signal through Nanog to modulate chemoresistance, EMT, immune evasion, and/or CSCs (Figure 1). Our current understanding of the Nanog signaling cascade is limited to data generated using a heterogeneous population of carcinoma cells. It is of high interest to determine if Nanog circuitry in CSCs is distinct or overlaps with the bulk non-tumorigenic carcinoma cells. This line of investigation may be difficult to perform from a technical prospective due to the limited number of CSCs and the transient state of CSCs. In any event, a systems biology approach will be needed to allow deeper understanding of the temporal and intensity dynamics of Nanog circuitry in carcinoma cells and CSCs. This information will be essential to identify critical “druggable” points in the Nanog circuitry in order to develop anti-Nanog therapeutics with optimal therapeutic efficacy and minimal off-target effects.

Figure 1. Schematic model of the Nanog signaling circuitry in epithelial carcinomas.

Arrow lines represent activation. Dot lines represent inactivation. All interactions are based on published literature as described in the text.