1
INDEX
ABBREVIATIONS 5
ABSTRACT 9
RESUMEN 11
1 INTRODUCTION 15
1.1 INFLAMMATION: A CAUSE OF CANCER DEVELOPMENT 15
1.1.1 COLORECTAL CANCER DEVELOPMENT 16
1.2 IMPLICATION OF COX2 AND PROSTAGLANDINS IN CANCER 17 1.2.1 PGE2 SIGNALING RELEVANCE IN EPITHELIAL OVARIAN CANCER 19 1.3 PMEPA1: PROSTATE TRANSMEMBRANE PROTEIN ANDROGEN INDUCED 1 20
1.3.1 PMEPA1 EXPRESSION IN CANCER 22
1.3.2 PMEPA1 IS AN ANDROGEN INDUCIBLE GENE 23
1.3.3 PMEPA1 IS A TRANSFORMING GROWTH FACTOR-β INDUCIBLE GENE 23
1.4 NUCLEAR FACTOR-κB IMPLICATION IN CANCER 29
1.4.1 ANTISENSE TRANSCRIPT TO PMEPA1: NKILA 30
2 OBJECTIVES 35
3 MATERIALS AND METHODS 39
3.1 MATERIALS 39
3.1.1 Cell lines 39
3.1.2 Reagents 39
3.1.3 Oligonucleotide primers used for quantitative real time PCR 40
3.1.4 Antibodies 42
3.1.5 Plasmids 43
3.2 METHODS 45
3.2.1 Plasmids cloning 45
3.2.2 Transient cell transfection 45
3.2.3 Lentiviral cDNA transfection 46
2
3.2.4 Cell proliferation assays 46
3.2.5 Cell migration assays 47
3.2.6 mRNA extraction and real time quantitative PCR (RT qPCR) 47
3.2.7 Immunofluorescence assays 47
3.2.8 Western blotting: cell lysates 48
3.2.9 Protein immunoprecipitation 49
3.2.10 RNA immunoprecipitation 49
3.2.11 Luciferase assays 50
3.2.12 Tumourigenesis in nude mice 50
3.2.13 Tumour extraction from mice 50
3.2.14 Histological analysis and immunohistochemistry 51
3.2.15 Statistical analysis 51
4 RESULTS 55
4.1 PMEPA1 EXPRESSION 55
4.1.1 Induction of PMEPA1 expression by COX2 and PGs 55
4.1.2 PMEPA1 induction by TGF-β 56
4.1.3 PMEPA1 induction by Ca2+ 56
4.1.4 Prostaglandin F2α – F prostanoid receptor signaling 56
4.2 IN VITRO PMEPA1 OVEREXPRESSION IN CANCER CELLS 59
4.2.1 PMEPA1 affects cell proliferation 60
4.2.2 SKOV3 undergo cell behaviour changes with PMEPA1 overexpression 61
4.3 MECHANISMS OF ACTION OF PMEPA1 63
4.3.1 PMEPA1 involvement in TGF-β signaling pathway 63 4.3.2 High levels of E-Cadherin protein in PMEPA1 overexpressing SKOV3 cells 64
4.3.3 PMEPA1 affects SMADs proteins 65
4.3.4 β-catenin localization is affected by PMEPA1 expression 66
3 4.3.5 PMEPA1 expression enhances P-GSK3β protein levels 67 4.3.6 PMEPA1 inhibits mesenchymal cell characteristics. 69 4.4 OVCAR8 CELLS SHOW SIMILAR BEHAVIOUR AS SKOV3 CELLS BY PMEPA1
OVEREXPRESSION 71
4.5 IN VIVO PMEPA1 FUNCTION IN ONCOGENESIS 73
4.5.1 Subcutaneous xenograft mouse model 73
4.5.2 Subcutaneous and intraperitoneal xenograft mouse model 75
4.5.3 Orthotopic xenograft mouse model 78
4.6 PMEPA1 EXPRESSION IN HUMAN OVARIAN TUMOURS AND PERITONEAL
METASTASES 80
4.7 PMEPA1 EXPRESSION IN TISSUE ARRAYS 83
4.8 DIFFERENTIAL ACTIVITY OF PMEPA1 ISOFORMS 85
4.8.1 Different transcripts of PMEPA1 have different functions 85 4.8.2 PMEPA1 transcripts are differently induced by TGF-β and Fluprostenol 86 4.8.3 β-catenin nuclear translocation is blocked by all PMEPA1 transcripts 88 4.8.4 PMEPA1 transcripts overexpression in SKOV3 cells affects the different
NKILA transcripts levels 89
4.8.5 PMEPA1 isoforms interact physically with IκB and NKILA 89 4.8.6 PMEPA1 transcripts amplify different CSC markers RNAs 90 4.8.7 NF-κB activation by PMEPA1 transcripts overexpression 91
5 DISCUSSION 95
6 CONCLUSSIONS 105
CONCLUSIONES 107
7 REFERENCES 111
8 APPENDIX 117
4
5
ABBREVIATIONS
µg microgram
µl microliter
AKT (PKB) Protein kinase B
AR Androgen receptor
BCA Bicinchoninic acid
bp Bases pairs
BSA Bovine serum albumin
Ca2+ Calcium
cDNA Complementary deoxyribonucleic acid COX2 Cyclooxygenase 2
CRC Colorectal cancer d.p.i. Days post-injection DNA Deoxyribonucleic acid
END Endothelin
ENG Endoglin
EOC Epithelial ovarian cancer EP (1-4) Prostaglandin receptors FBS Fetal Bovine Serum FP Prostaglandin F receptor
GSK-3αβ Glycogen synthase kinase3 alpha/beta IBD Inflammatory bowel disease
ID-1 Inhibitor of differentiation/DNA binding 1
IKK IκB kinase
IκBα Inhibitor of NF-κB alpha protein lncRNAs Long non coding RNAs
LPS Lipopolysaccharide
MEM Minimum essential medium
mPGES Microsomal prostaglandin E synthase mRNA Messenger ribonucleic acid
NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells
6
NFκBIA NF-κB inhibitor alpha gene
NSAIDs Nonsteroidal anti-inflammatory drugs OD Optical Density
PAI-1 Plasminogen activator inhibitor-1 PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PGDS Prostaglandin D synthase PGE2 Prostaglandin E2
PGES Prostaglandin E synthase PGF2α Prostaglandin F2α
PGIS Prostaglandin I synthase PGs Prostaglandins
PTEN Phosphatase and tensin homolog RNA Ribonucleic acid
RT Room temperature
RT-qPCR Reverse transcription –quantitative PCR TGF-β Transforming growth factor beta TGFβRI TGF-β receptor type I
TGFβRII TGF-β receptor type II TNFα Tumour necrosis factor alpha TXS Thromboxane synthase
VEGF Vascular endothelial growth factor
WB Western Blot
7
A BSTRACT
8
9
ABSTRACT
Cyclooxygenase 2 (COX2) elevated expression has been associated with tumour development especially in colorectal cancer and it is well established that COX2 inhibition can help prevent cancer. In order to study the mechanisms that mediate the pro-tumourigenic effects of COX2, cell lines stably overexpressing this gene were generated and gene expression microarray analysis revealed that PMEPA1 was one of the up-regulated genes. It was originally identified as an androgen-induced gene with abundant expression in several cancers. The encoded product is a transforming growth factor-β (TGFβ)-induced transmembrane protein overexpressed in breast, prostate and colorectal cancer. We also found that PMEPA1 can be upregulated by COX2 overexpression in colon cancer cells and the prostaglandins produced by them.
In view of these observations, we overexpressed PMEPA1 in human colon adenocarcinoma (HT29) and human ovarian carcinoma (SKOV3) cells to test if part of the protumourigenic effects of COX2 were due to PMEPA1. PMEPA1 overexpression increased cell proliferation rates. Also, PMEPA1 provided a negative feedback loop over TGF-β signaling, regulating SMAD and non- canonical signalling pathways. The actin cytoskeleton distribution was modified in PMEPA1 overexpressing SKOV3 cells, resulting in differences in cell morphology. Immunofluorescence revealed β-catenin nuclear translocation blockade when PMEPA1 is overexpressed. Additionally, subcutaneous and intraperitoneal mouse xenografts experiments in nude mice showed a differential effect of PMEPA1 overexpressing HT29 and SKOV3 cells, leading to a final growth delay of HT29 xenografts, while PMEPA1 overexpressing SKOV3 cells have a high survival and proliferative advantage. In addition, orthotopic mouse xenograft experiments, with SKOV3 cells, showed the same proliferative and establishment advantage when PMEPA1 is overexpressed.
Different PMEPA1 transcripts have been described, that differently overlap with NKILA sequence (a lncRNA that regulates NF-κB). We found that each transcript differently coprecipitates with IκBα and with NKILA and thus regulates signaling of NF-κB and SMAD pathways.
Finally, immunohistochemistry of human tumour samples revealed a high PMEPA1 expression in patients with advanced tumour stages. These findings suggest that PMEPA1 may have an important role in cancer progression, and could be a diagnostic and prognostic biomarker in colorectal and ovarian tumours, but further investigations should be done.
10
11
RESUMEN
La elevada expresión de la Ciclooxigenasa 2 (COX2) ha sido asociada con el desarrollo tumoral especialmente en cáncer colorrectal y está bien establecido que la inhibición de COX2 ayuda a prevenir y tratar el cáncer. Con el objetivo de estudiar los mecanismos de los efectos tumorigénicos de COX2, se generaron líneas celulares que sobreexpresaban este gen y su análisis reveló que PMEPA1 era uno de los genes inducidos por COX2. PMEPA1 ha sido identificado previamente como un gen inducido por andrógenos y con alta expresión en varios tipos de cáncer. El producto codificante es una proteína transmembrana inducida por el factor de crecimiento transformante-β (TGF-β), sobreexpresada en cáncer de mama, próstata y colorrectal principalmente.
Observamos que la expresión de PMEPA1 podía estar inducida por la sobreexpresión de COX2 y por las prostaglandinas producidas. Para analizar su efecto, PMEPA1 fue sobreexpresada en células de adenocarcinoma de colon (HT29) y de carcinoma de ovario (SKOV3) humanos para comprobar si parte de los efectos pro-tumorigénicos de COX2 son debidos a PMEPA1. La sobreexpresión de PMEPA1 incrementaba el crecimiento celular.
Además, PMEPA1 ejerce una regulación negativa sobre TGF-β, regulando la señalización de las proteínas SMAD y de la vía no canónica. La distribución del citoesqueleto de actina se ve modificada en las células SKOV3 que sobreexpresan PMEPA1, que resulta en diferencias en la morfología celular. Por ello, las inmunofluorescencias revelaron que PMEPA1 bloquea la translocación nuclear de β-catenina. En paralelo, los ensayos de xenoinjertos subcutáneos e intraperitoneales con ratones nude, mostraron diferencias entre las células HT29 y SKOV3 que sobreexpresan PMEPA1, con una gran ventaja proliferativa y de supervivencia en las SKOV3 respecto de las HT29. Los modelos de xenoinjertos ortotópicos de ovario mostraron la misma ventaja proliferativa y de establecimiento celular de las SKOV3 que sobreexpresan PMEPA1.
PMEPA1 tiene diferentes tránscritos que se superponen de forma diferente con la secuencia de NKILA (un RNA largo no codificante). Hemos comprobado que los distintos tránscritos co-precipitan con IκBα y NKILA de forma distinta y, por lo tanto, regulan la señalización de NF-κB y SMADs.
Finalmente, las inmunohistoquímicas en muestras de tumores humanos han revelado la alta expresión de PMEPA1 en pacientes con estadíos tumorales avanzados. Por todo ello, PMEPA1 podría tener un papel importante en la progresión del cáncer y podría ser un biomarcador de diagnóstico y pronóstico en tumores colorrectales y de ovario.
12
13
I NTRODUCTION
14
15
1 INTRODUCTION
1.1 INFLAMMATION:ACAUSEOFCANCERDEVELOPMENT
Cancer has emerged as a leading cause of morbidity and mortality in European populations, causing seven million deaths annually, accounting for 12.5% of deaths worldwide, being the second leading cause of death in the developed world and is among the three leading causes of death for adults in developed countries. The number of new cases is expected to rise by about 70% over the next 2 decades, with already approximately 14 million new cases in 2012.
(Stewart and Wild 2014). In Spain, cancer is the first death cause and among this maladies group, pulmonary, colon and breast cancer are the main mortality and morbidity causes Smoking, dietary habits and reproductive factors have been identified as the main (modifiable) risk factors for cancer in populations of industrialized countries, with different implications across countries and regions (Arnold et al. 2015).
The functional relationship between inflammation and cancer is not new: already in 1863, Virchow hypothesized that the origin of cancer was at sites of chronic inflammation. He observed “lymphoreticular infiltrate” in neoplastic tissues that reflected the origin of cancer at sites of chronic inflammation and made a connection between inflammation and cancer. Today, the causal relationship between inflammation, innate immunity and cancer is widely accepted;
however, many of the molecular and cellular mechanisms participating in this relationship remain unresolved (Grivennikov et al. 2010).
A hypothesis of how inflammatory cells cooperate in the neoplastic process is that many malignancies arise from areas of infection and inflammation, just as part of the normal host response. Indeed, constant infections within the host induce chronic inflammation.
Inflammatory cells, as leukocytes and other phagocytic cells (as part of the immune response), induce DNA damage in proliferating cells through generation of reactive oxygen and nitrogen species that are produced normally by these cells to fight infection. These products interact with DNA and could result in permanent genomic alterations such as point mutations, deletions, or rearrangements. Actually, for example, p53 mutations are detected in chronic inflammatory diseases, as well as in tumours, such as rheumatoid arthritis and inflammatory bowel disease (IBD) (Madanchi et al. 2016).
16
INTRODUCTION
1.1.1 COLORECTAL CANCER DEVELOPMENT
IBD are inflammatory disorders of the intestinal tract that are most common in developed countries, affecting the quality of life of approximately 1.4 million individuals in the United States and 2.2 million in Europe. Epidemiological and clinical studies indicate that patients affected by the two major forms of IBD (ulcerative colitis and Crohn’s disease) have an increased risk of developing colorectal cancer (CRC). CRC is the most common malignancy and the second most frequent cancer death cause in Europe and Spain -more than 13000 deaths per year in Spain- (Ferlay et al. 2010) (Figure 1). Even in patients who have undergone tumour resection, 40–50% relapse and die of metastases, being the overall 5-year survival less than 60% (Kang et al. 2005). These statistics show that therapeutic options nowadays available do not reach a desirable efficacy, so new therapeutic strategies are necessary for established tumours, as well as preventive strategies.
Fig.1 Trends in colon cancer incidence in males (left panel) and female (right panel) by country and region 1988-2008 (M. Arnold et al. 2015)
Multiple data indicates that human cancer is a multistage genetic and epigenetic disease. CRC, as all cancers, are thought to be originated from a single replication-competent cell (stem cell or proliferative progenitor cell). Colorectal tumours could be initiated by the acquisition of a genetic alteration that deregulates the ‘gatekeeping’ pathway—the
17 INTRODUCTION
adenomatous polyposis coli (APC)/β-catenin pathway (also known as the WNT-signaling pathway)—with tumour progression occurring through clonal selection with acquisition of multiple genetic lesions (Vogelstein and Kinzler 2004). These alterations have the potential to produce different cancer cell genotypes; however, have been proposed to affect six principal aspects of cell physiology that are essential for cancer development. These are termed the
‘hallmarks of cancer’: acquired capabilities that represent breaches to the normal regulatory mechanisms controlling cell survival, proliferation, migration, invasion and the interactions with neighboring cells and stroma.
In CRC, the genetic changes that occur follow the histological progression from small, pre-malignant adenomas, to advanced metastatic tumours, with both early and late stages of the disease associated with the number of sequential genetic alterations. Alterations in three types of genes are responsible for tumourigenesis: oncogenes, tumour-suppressor genes and stability genes. Each genetic event confers an advantageous characteristic upon the expanding tumour mass. While it may be true that numerous different genes can become altered during the development of a tumour, it has also been proposed that all cancers arise and are maintained by the deregulation of a relatively small number of signaling pathways (Armaghany et al. 2012).
Deregulation of the WNT-signaling pathway is believed to initiate colorectal tumours, otherwise, the aberrant expression of COX2 that occurs in the majority of colorectal tumours has been demonstrated to play a crucial role during CRC development.
1.2 IMPLICATIONOFCOX2ANDPROSTAGLANDINSINCANCER
Clinical trials have shown that non-steroidal anti-inflammatory drugs (NSAIDs) treatments reduce the risk of developing cancer, and more specifically CRC (Mizuno, S. et al. 2006). NSAIDs inhibit the cyclooxygenase (COX) enzymatic activity, so it could be a mechanism of cancer prevention. There are two enzymes with cyclooxygenase activity: COX1 (PTGS1) constitutively expressed in most tissues maintaining homeostasis, while COX2 (PTGS2) is induced by various stimuli, including cytokines, growth factors, hormones, tumour promoters and hypoxia (Smith et al. 1991) COX2 has a differential sensitivity to NSAID inhibition than COX1 enzyme. COX2 expression has been detected in different tumours, including CRC, which could explain the NSAIDs beneficial effects in cancer prevention and treatment (Rizzo 2011).
18
INTRODUCTION
COX are the enzymes of the rate limiting step in the biosynthesis of prostaglandins (PGs).
There are five primary prostanoids: prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2a (PGF2a), prostaglandin I2/ prostacyclin (PGI2), and thromboxane A2 (TXA2).
Their biosynthesis consist of a three-step sequence: release of Arachidonic Acid (AA) from phospholipids by phospholipases A2 (PLA2); bis-oxygenation of AA, yielding the prostaglandin endoperoxide PGG2, which is reduced to the prostaglandin endoperoxide PGH2; and the conversion of PGH2 to the final metabolites PGD2, PGE2, PGF2, PGI2, or TXA2 via specific synthases or isomerases (Figure 2). We have described that the respective synthases for PGE2, PGD2, PGF2α
and PGI2 are present in the healthy colon as well as in CRC (Stamatakis et al. 2015). COX-derived prostanoids are active lipid mediators involved in a range of physiological processes such as modulation of vascular tone, the inflammatory response and gastric cytoprotection, they have also been implicated in various disease states such as arthritis, heart disease and pulmonary hypertension (Iniguez et al. 2008; Iñiguez et al. 2003).
Furthermore, in cancer studies, prostanoids have been shown to regulate angiogenesis in carcinoma and chronic inflammatory disease progression (Cathcart et al. 2012). Little is known about how prostanoids mediate tumour-induced angiogenesis, although prostanoid signaling has been analyzed in inflammation.
Fig.2 Arachidonic acid (AA) cascade. The conversion of AA to prostaglandins and thromboxane through COX2 and COX-1 enzymatic activity. Each prostaglandin with its receptor is represented.
TXS
PGDS PGIS PGES PGFS
19 INTRODUCTION
Deregulation of COX2 expression leads to an increased abundance of its principal metabolic product, prostaglandin E2 (PGE2), and the best studied in the context of tumour progression, which pleiotropic effects appear to affect most of the hallmarks of cancer.
The pro-tumourigenic effects of COX2 in the colon have been attributed to its role in producing PGE2, which is reported both in human colorectal adenomas and carcinomas. PGE2
levels seem to increase in a size-dependent manner in the adenomas of familial adenomatous polyposis patients. COX2/PGE2 pathway affects multiple aspects of cell physiology required for tumour development and maintenance (Salvado et al. 2012).
COX2 participates in tumour development in different ways: increases proliferation, decreases apoptosis, induces tumour angiogenesis and metastasis (Iñiguez et al. 2003; Rizzo 2011). The proangiogenic effects of COX2 can be inhibited by NSAIDs, resulting in the inhibition of endothelial cell proliferation, migration and vascular tube formation (Suri et al. 2016).
Few progresses in the use of drugs in prevention of cancer had taken place. COX2 inhibitors may be such drugs. In this regard, several lines of evidence suggest that long-term use of aspirin might reduce the risk of some cancers, particularly gastrointestinal tumours.
Aspirin studies have shown how it reduces incidence or growth rate of several cancers in animal models mediated in part by inhibition of COX enzymes and reduced production of prostaglandins and other inflammatory mediators (Rothwell et al. 2011).
In the last years, considerable interest has aroused in targeting downstream effectors of the COX signaling pathway in cancer. Selective targeting of these downstream effectors could have the potential of avoiding the cardiovascular effects associated with selective COX2 inhibition, while maintaining anti-cancer properties (Cathcart et al. 2012).
1.2.1 PGE2 SIGNALING RELEVANCE IN EPITHELIAL OVARIAN CANCER
Epithelial ovarian cancer (EOC), which comprises 90% of all ovarian malignancies, is the leading cause of death from gynecological cancer in developed countries since the majority of patients present disseminated disease at diagnosis, for which the average 5-year survival rate is low (Jemal et al. 2006). The cell and stage specific increases of COX1, COX2, mPGES-1 and EP1–
2 in EOC support the hypothesis that PGE2 synthesis and signaling are of importance for malignant transformation and progression in EOC, which have implications for future therapies (Rask et al. 2006).
Furthermore, mPGES-1 and the EP receptors may represent important targets for development of novel anti-inflammatory and antitumour therapies, as EP2 receptor has been
20
INTRODUCTION
described to have a key role in the ovulatory process (Rask et al. 2006). Increased contents of PGE2 in ovarian tumours have been described in previous studies (Ali-Fehmi et al. 2005), possibly regulating cell proliferation and apoptosis in ovarian cancer (Munkarah et al. 2002).
In view of these results, and as the use of NSAIDs or aspirin have demonstrated to reduce the risk to develop CRC and in a lower level in prostate and breast cancer, ovarian cancer development could also be prevented by COX2 inhibition (Baandrup et al. 2013). As an example, it has been described that Celecoxib, a selective COX2 inhibitor is a potent inhibitor of ovarian cancer cell growth in vitro and in vivo under different metabolic conditions likely by reducing tumour angiogenesis (Suri et al. 2016).
Previous studies have determined whether COX2 mediates epithelial growth factor (EGF)- induced cell invasion in human ovarian cancer cells, and the overexpression of EGF receptor (EGFR) in human ovarian cancer is associated with poor prognosis and disease progression. EGF induces human ovarian cancer cell invasion by downregulating the expression of E-cadherin through various signaling pathways (Cheng et al. 2012). EGF treatment can increase COX2 expression and PGE2 production in human ovarian cancer cell lines; this EGF-induced COX2 expression is mediated by the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway.
Moreover, inhibition of COX2 and PGE2 attenuates EGF-induced cancer cell invasion (Qiu et al. 2014). Thus, all these results demonstrate that COX2 and PGE2 are involved in EGF-induced human ovarian cancer cell invasion and development that should be deeply studied for future therapies development and to find new biomarkers.
1.3 PMEPA1:PROSTATETRANSMEMBRANEPROTEINANDROGENINDUCED1
A few years ago, a novel gene was identified being upregulated in renal cell carcinoma and was designated as solid tumour associated gene 1 (STAG1) (Rae et al. 2001). STAG1 also called PMEPA1/TMEPA1 is a prostate transmembrane protein androgen induced 1 gene, located on the chromosome region 20q13.31, a region frequently amplified in multiple solid tumours, such as prostate, colon, breast and ovarian cancers (R. Liu et al. 2011) (Figure 3). ERG1.2 is the mouse homolog of PMEPA1, one of the EGF pathway target genes, abnormally activated and common in human cancer (Giannini et al. 2003).
21 INTRODUCTION
Chromosome 20 - NC_000020.11
Fig.3. PMEPA1 and other genes expression in chromosome 20, Assembly: GRCh38.p10 Location:
chr20:64,095,055-64,274,842. Cytogenetic location: 20q13.3 (Genome Reference Consortium)
The complete sequence of STAG1 contained 320 bp of 5’ untranslated region (UTR) and a 3’ UTR of 3658 bp, with the largest open reading frame of 861 bp (nucleotides 321 ± 1181), and encoded a 287 aminoacid protein of 32 kDa approximately. Consensus polyadenylation signals (AATAAA) were located at nucleotides 2158, 2463, and 4818 and it contains three methionine codons, which could function as initiation translation sites. The STAG1 cDNA encompassed 1042 nucleotides (nucleotides 430 ± 1458) of the reported PMEPA1 cDNA (L L Xu et al. 2000), that is why it began to be referred to as STAG1/PMEPA1. This gene also showed homology (77%), at the nucleotide level, over the coding region of chromosome 18 open reading frame 1 (C18orf1) cDNA (Rae et al. 2001).
It has been also described that PMEPA1 has a 5’ non-coding region of 320 pb and a 3’
non-coding region of 3658 bp. PMEPA1-encoded protein is a type Ib transmembrane protein that shows high sequence homology to a mouse N4wbp4-encoded protein that binds to NEDD4 protein, an E3 ubiquitin–protein ligase involved in ubiquitin-dependent, proteasome-mediated protein degradation (Linda L. Xu et al. 2003). PMEPA1 contains the transmembrane domain (from 41 to 62 amino acids) at the extracellular amino terminal (N-terminus) end while the carboxyl end is intracellular (Rae et al. 2001). It also contains two PY motifs (PPPY and PPTY) which are required for binding to WW domains of NEDD4 E3 ubiquitin ligase (Luo et al. 2017).
NEDD4 (isoform 1) may function as an oncogene by reducing cytoplasmic levels of PTEN in cancer cells and plays a direct role in reducing the levels of RNA polymerase II during cellular damage responses. Therefore, NEDD4-interacting proteins may play significant roles in controlling the availability of NEDD4 in cancer cells.
On the other hand, different PMEPA1 alternative splicing variants have been described, in which the transmembrane domain is lost and the protein has a different subcellular localization (Brunschwig et al. 2003). Further studies should be done to understand each PMEPA1 variant function and localization.
22
INTRODUCTION
1.3.1 PMEPA1 EXPRESSION IN CANCER
Databases analyses showed that PMEPA1 is expressed in pancreatic, endometrial, and prostatic adenocarcinomas (Brunschwig et al. 2003), in CRC primary tumours and metastasis (Vo Nguyen et al. 2014) and in breast cancer (Singha et al. 2010, 2014). Previous studies have also revealed a high PMEPA1 expression in prostate and uterus healthy tissues (L L Xu et al. 2000).
However, high PMEPA1 gene expression is found in 87% in renal cancer cells (RCC) and in samples from stomach and rectum adenocarcinomas; contrary, its expression was hardly detectable in leukemia and lymphoma samples. PMEPA1 high expression in different cancer types could be due to the gene localization in the 20q13 chromosome frequently amplified in different types of cancer, as in colon, breast, and ovarian cancers (Singha et al. 2014).
Studies of PMEPA1 (Rae et al. 2001; L L Xu et al. 2000; Linda L. Xu et al. 2003) in cell growth regulation have revealed elevated PMEPA1 expression in non-tumourigenic revertants of tumour cell lines and alterations of PMEPA1 expression (either up- or down-regulation) in human tumours. Opposite PMEPA1 effects on cell growth have been published, as cell proliferation seems to be inhibited by STAG1/PMEPA1 overexpression in prostate cancer cells, while a tumour prostate subcellular isolated clone expresses high level of PMEPA1 with rapid growth in nude mice (Hirokawa et al. 2007). The role of STAG1/PMEPA1 in the context of cellular proliferation of cell lines of different tumour origins should be elucidated.
Furthermore, in prostate cancer, DNA methylation could play a role in downregulating PMEPA1 gene transcription depending on androgen receptor expression on prostate cell lines (Richter et al. 2007), while PMEPA1 has been described as a gene that lacks specificity for detecting prostate epithelial cells (Cardillo, I, and Bracci 2006). In other cancers, such as esophageal adenocarcinoma, PMEPA1 stromal signature has been studied and its overexpression has a significantly poorer outcome in those patients (Saadi et al. 2010).
PMEPA1 has also been suggested as a novel transcriptional target for p53 (the most frequently mutated gene in human tumours), which could mediate p53-dependent apoptosis, and has been considered as a good candidate for future gene therapy (Anazawa et al. 2004).
The high PMEPA1 expression in different tumours and the potential of PMEPA1 protein as a NEDD4-binding partner (Seyhan et al. 2011) suggested cell growth regulatory functions of PMEPA1 and have provided the impulse to study the cell biological roles of PMEPA1.
23 INTRODUCTION
1.3.2 PMEPA1 IS AN ANDROGEN INDUCIBLE GENE
PMEPA1 was identified originally as a highly androgen-inducible gene with abundant prostate expression that was restricted to prostatic epithelial cells, but finally it has been described as a direct transcriptional target of the androgen receptor (AR) (Mcmaster et al. 2010) that negatively regulates AR protein levels (Li et al. 2008). The biologic effects of androgen on target cells are mediated, in part, by transcriptional regulation of androgen-regulated genes.
Alterations of the AR structure or function can affect the normal transcriptional regulation of the androgen-regulated genes, which may contribute to the process of prostate cancer initiation and progression. Different studies of multiple prostate tissues suggest the high level of PMEPA1 expression in prostate (Li et al. 2008; Linda L. Xu et al. 2003); this expression has been predominately detected in glandular epithelial prostate cells.
PMEPA1-dependent down-regulation of AR has been described as a cause of AR ubiquitination and proteasome-mediated degradation, probably by recruitment of NEDD4 protein (Li et al. 2008), while there are other mechanisms of AR ubiquitination and degradation pathways linked to the NEDD4 E3 ubiquitin ligase. AR and PMEPA1 are thought to form a feedback loop, and frequent loss of PMEPA1 expression noted in prostate tumours may lead to the gain of AR function contributing to prostate cancer.
Both studies previously mentioned indicate that PMEPA1 overexpression leads to cell growth inhibition in prostate cancer, while its expression decreased during prostate cancer progression. Nevertheless, these studies are focused only in prostate cancer analysis, while PMEPA1’s role in other types of cancer remains to be further studied.
1.3.3 PMEPA1 IS A TRANSFORMING GROWTH FACTOR-β INDUCIBLE GENE
PMEPA1 is known to be induced by transforming growth factor-β (TGF-β) (Brunschwig et al. 2003). TGF-β is a ubiquitous cytokine that plays a critical role in numerous pathways regulating cellular and tissue homeostasis. It is involved in differentiation, migration, proliferation and adhesion. This cytokine exhibits tumor suppressor and pro-tumorigenic and pro-metastatic actions, depending on the stage of cancer development and the specific cell type within the tumor micro-environment (Caja et al. 2012). Thus, TGF-β could act as a tumour suppressor by inhibiting cell growth, but it also promotes tumour progression and metastasis by inducing epithelial to mesenchymal transition, invasion, immunosuppression, and blood vessel intravasation by carcinoma cells.
24
INTRODUCTION
The effects of TGF-β and related factors depends on the state of responsiveness of the target cell as on the factors themselves. The TGF-β family comprises a large number of structurally related polypeptide growth factors, each capable of regulating different cellular processes (Massagué 1998). The TGF-β family is mainly composed by the isoforms TGF-β1, TGF- β2 and TGF-β3, by bone morphogenetic proteins (BMP) and Activin subfamilies. BMPs are the extracellular signaling molecules that represent the largest subgroup of this TGF-β superfamily of proteins. BMP4 is one of the most studied BMP members, having gene variants that predispose to CRC and its expression levels are altered in many tumor types and linked to patient prognosis in ovarian cancer (Kallioniemi et al. 2012).
TGF-β signaling inhibition is one of PMEPA1’s roles described in several studies. Because TGF-β mediates an apoptotic pathway in colon cancer, PMEPA1 overexpression is stipulated to promote cancer cell growth by forestalling apoptosis (Sheffer et al. 2009). PMEPA1 also has been mentioned as responsible to convert TGF-β from a tumour suppressor to a tumour promoter in breast cancer (Singha et al. 2010) and, in addition, TGF-β-dependent growth of aggressive breast cancer has been suggested to depend on increased expression of PMEPA1 gene (Singha et al.
2014). Moreover, PMEPA1 has been described as a TGF-β direct target gene which induction doesn’t need any intermediate protein (Brunschwig et al. 2003). PMEPA1 induction by TGF-β has been reported as a negative feedback loop, up-regulating the proto-oncogene c-Myc (PMEPA1 depletion decreased the protein expression levels of c-Myc, a well-known TGF-β- SMAD3 target that inhibits p21 gene transcription) and down-regulating p21 (SMAD3/4 complex is essential for endogenous PMEPA1 to suppress p21 expression through c-Myc), a regulator of cell cycle, having finally cell cycle promotion (R. Liu et al. 2011).
1.3.3.1 CANONICAL AND NON-CANONICAL TGF-β SIGNALING PATHWAYS
Different protein pathways are involved in TGF-β signaling pathways, named the canonical and non-canonical cascades. TGF-β and related factors signal through a family of transmembrane protein serine/threonine kinases referred to as the TGF-β receptor family, which is divided into two subfamilies: type I receptors (TGFβRI) and type II receptors (TGFβRII) based on their structural and functional properties. In the absence of ligand, type II and type I receptors exist as homodimers at the cell surface (Derynck and Zhang 2003). For type I receptors, the neutral nomenclature ALK (activin receptor–like kinase) has been used; thus, the TGFβRI (the classical TGF-β receptor) is originally known as ALK5. It has been described that ALK1 (another type I receptor) binds TGF-β but does so more weakly than ALK5 and is not known to mediate a TGF-β response (Cheifetz et al. 1987). Additionally, combinatorial interactions in the
25 INTRODUCTION
tetrameric receptor complex allow differential ligand binding or differential signaling in response to the same ligand.
The TGF-β/SMAD or canonical signaling pathway, begins with the binding of a TGF-β family member (which was previously attached in the extracellular matrix to latent TGF-β binding proteins -LTBPs) to the TGF-β receptor II (TGFβRII) that activates by phosphorylation a TGF-β receptor I (TGFβRI), leading to formation of a receptor complex and phosphorylation of the type I receptor (Lampropoulos et al. 2012). Once the type I receptor is activated, it subsequently phosphorylates a receptor-SMAD (R-SMAD), which forms a heterotrimeric complex of two R-SMADs and SMAD4, and translocate into the nucleus (Watanabe et al. 2010) (Figure 4). Among R-SMADs, SMAD2 and SMAD3 act downstream of TGF-β, activin, and nodal type I receptors, whereas SMAD1, SMAD5, and SMAD8 are phosphorylated by BMP type I receptors. All these SMAD proteins exist as homo-oligomers in the cytoplasm, in a basal state.
Fig.4 Scheme of TGF-β/SMAD signaling pathway. SMAD 2/3 and SMAD4 complex translocate to the nucleus to initiate gene transcription and subsequent different effects on cell behavior (Diagram adapted from Derynck, R., & Zhang, Y. E. 2003).
26
INTRODUCTION
Once the SMAD complex is in the nucleus, it associates with a DNA-binding partner (as CAGA box) and this complex binds to specific enhancers, elements controlling expression of target genes. Human plasminogen activator inhibitor-1 (PAI-1) is a gene that is potently induced by TGFβ, due to SMAD3/SMAD4 binding sequences, termed CAGA boxes, within the promoter of the gene (Dennler et al. 1998). Endothelin-1 (ET-1) has also been described as a SMAD 3/4 dependent gene (Rodríguez-Pascual et al. 2004). However, other target genes, as ID1, are mainly induced by the BMP signaling pathway (Ying et al. 2003).
Three variants of the human PMEPA1 gene have been registered in gene bank. Two variants have a transmembrane domain (TM), whereas the third does not. These three human variants blocked TGF-β activity and SMAD2 phosphorylation. The variant deficient in TM domain could also inhibit the TGF-β signal. Thus, the integration of PMEPA1 in the membrane is not required for its inhibitory effect on TGF-β signaling (Watanabe et al. 2010).
Previous studies have shown that PMEPA1 localizes to the lysosome and downregulates TGF-β signaling by competing with SARA (SMAD anchor for receptor activation) for R-SMAD binding to sequester R-SMAD phosphorylation and promoting lysosomal degradation of TGF-β receptor (Watanabe et al. 2010). So, PMEPA1 could decrease growth suppressive signaling induced by TGF-β, suggesting that its overexpression and/or increased or altered function may be a ‘molecular switch’ that converts TGF-β from a tumour suppressor to a tumour promoter.
Still, TGF-β is involved in many molecular pathways. On the other hand, TGF-β dependent tumour promoting phosphatidylinositol 3-kinase/ protein kinase B (PI3K/Akt) or the non-canonical signaling pathway, is increased by elevated expression of PMEPA1.
Activation of PI3K/Akt cascade, which promotes cancer formation and growth, could also be achieved through a variety of mechanisms as inactivation of PTEN (phosphatase and tensin homolog), a lipid phosphatase that antagonizes PI3K/Akt signaling. Accordingly, endometrial cancer is typified by alterations in PI3K/Akt signaling, which includes inactivating mutations in PTEN among others. Furthermore, the genetic deletion of murine Nedd4 results in a strong inhibition of the PI3K/Akt signaling (Zhang et al. 2015). Tumour suppression by TGF-β is abolished in several late stage cancers that become TGF-β dependent for growth and/ or metastasis, this non-canonical signaling can be SMAD-independent, but often needs SMAD- dependent inputs (Hoover et al. 2008).
The oncogenic serine/threonine kinase Akt1 (also known as PKBα) is a downstream effector of the PI3K frequently activated in human cancer. Related Akt2 gene is overexpressed in ovarian, pancreatic, breast, and follicular thyroid carcinomas. In ovarian cancer, Akt2 kinase
27 INTRODUCTION
high activity, amplification and overexpression are associated with aggressive clinical behavior, suggesting that Akt contributes to tumour progression (Grille et al. 2003). Akt2 is a crucial factor in the PI3K/Akt signaling pathway, as it regulates cell proliferation, differentiation and survival in physiological and pathological processes (W. Xu, Yang, and Lu 2015).
Glycogen synthase kinase-3 beta (GSK-3β) is a ubiquitously expressed serine/threonine kinase. GSK-3β is active in resting epithelial cells and can be inactivated by various signaling events, as the PI3K/Akt, the Wnt signaling, and extracellular signal-regulated kinase (ERK1/2/MAPK pathways). GSK-3β activity is necessary to maintain the epithelial architecture;
inhibition of its activity results in upregulation of Snail, leading to epithelial-to-mesenchymal transition (EMT) (Zheng et al. 2013).
The PI3K/Akt pathway positively regulates Wnt/ β-catenin in 2 different manners, both of which contribute to the induction of EMT. The first mechanism consists in the movement of β-catenin into the nucleus, typically accompanied by Akt phosphorylation. While in the second mechanism, GSK-3β induces the degradation of β-catenin and Snail. In conclusion, the activation of Akt increases intracellular β-catenin levels (W. Xu, Yang, and Lu 2015). Thus, GSK-3β activity (phosphorylation and dephosphorylation) should be studied to understand the mechanisms of different tumour cells that undergo EMT.
1.3.3.2 WNT/ β-CATENIN SIGNALING PATHWAY
PMEPA1 has also been described as a Wnt target gene, which aberrant activation could run to neoplasia (Reichling et al. 2005). Previous immunohistochemistry studies with ApcMin/+
mice developing multiple intestinal adenoma have demonstrated that PMEPA1 is expressed in highly proliferative adenoma with activated Wnt signaling (Watanabe et al. 2010).
TGF-β signaling blockade by PMEPA1 has been supposed to be the reason of PMEPA1 involvement in tumourigenicity, but how TGF-β and Wnt signals affect the activation of PMEPA1 gene is not well understood. Transcriptional cooperation between TGF-β and Wnt signaling could be affecting PMEPA1 gene regulation.
The binding of individual Wnt ligands to their receptor complex components activates the canonical Wnt/ β-catenin cascade. Therefore, this pathway controls the intracellular levels of β-catenin. In the absence of Wnt signals, free β-catenin is targeted by a cytoplasmic protein complex (the β-catenin destruction complex), which promotes its phosphorylation, ubiquitination and degradation by the proteasome (Larriba et al. 2013). The stabilized β-catenin is maintained upon the activation of the canonical Wnt pathway and can be accumulated to the
28
INTRODUCTION
nucleus, forming an active transcription complex. This canonical Wnt/ β-catenin pathway collaborates with either TGF-β or BMP signaling in an agonistic or antagonistic manner. In an agonistic way, the nuclear complex of β-catenin interacts with SMAD proteins, whereas the transcript of the Id1 gene induced by BMP is negatively regulated by Wnt3a, which can inhibit the Id1 gene transcriptional complex formation (Nakano et al. 2010). β-catenin combines with intracellular domain of epithelial-cadherin (E-Cadherin) to form a complex. This complex subsequently connects to the actin cytoskeleton, mediates intercellular adhesion, and regulates tumor cell invasion and metastasis (W. Xu, Yang, and Lu 2015). The tight junctions formed between cells are constructed upon homotypic binding of E-cadherin.
Thus, PMEPA1, as a direct target gene for TGF-β signaling, is involved in the negative feedback loop of TGF-β signaling and might be one of the canonical Wnt target genes that could interact with either SMAD2 or SMAD3 via its SMAD interaction motif, blocking the TGF-β/SMAD signaling.
1.3.3.3 EPITHELIAL-TO-MESENCHYMAL AND MESENCHYMAL-TO-EPITHELIAL TRANSITION Epithelial-to-mesenchymal transition (EMT) is involved in the step of acquisition of migratory and invasive capability of cancer and the reverse process is called the mesenchymal- to-epithelial transition (MET). Cells with enhanced migratory/invasive properties are likely to contribute to tumour invasion and metastatic spread. EMT could occur in the primary tumours, cells enhance their ability to intravasation and generate circulating tumour cells. Once cells are extravasated, MET process would occur to favor the metastatic growth in secondary organs (Bonnomet et al. 2011). MET is also involved in colorectal carcinogenesis and it is essential for normal tissue and organ development. Therapeutically, the influence of EMT and MET is recognized, but the exact description of these biological phenomena is still not fully understood.
It is well established that E-cadherin expression and decreased cell mobility are common epithelial cell characteristics, while upregulation of N-cadherin, vimentin and zinc-finger domain proteins (SNAI1/SNAIL, SNAI2/SLUG), among others, are often linked to a mesenchymal-like phenotype. Cells that suffer EMT/MET exhibit important shape changes during which they can lose many of their epithelial or mesenchymal characteristics (Wells et al. 2008). A remarkable case is the ovarian surface epithelial cells, in which overexpression of E-cadherin induce a number of epithelial characteristics and markers associated with malignant transformation and tumour progression. Both primary and metastatic ovarian carcinomas express E-cadherin, in contrast to normal ovarian surface epithelium, which rarely expresses E-cadherin (Eriksson et al.
2006).
29 INTRODUCTION
Several oncogenic pathways (i.e., Wnt/β-catenin…) induce EMT. TGF-β is a potent inducer of this transition, directly activating the expression of transcription factors such as SNAI1/2, Twist and ZEB1/2, which are the key regulators of the EMT program. Related to this, SMADs (SMAD2 and SMAD3 particularly) have been proposed as critical mediators in EMT (Sipos and Galamb 2012). In view of this data, and because PMEPA1 is described as SMAD2/3 interacting protein, affecting TGF-β target genes transcription, the implication of PMEPA1 in the process of EMT/MET is worth to be studied.
1.4 NUCLEARFACTOR-ΚBIMPLICATIONINCANCER
Nuclear factor-κB (NF-κB) is a family of transcription factors that play critical roles in inflammation, immunity, cell proliferation, differentiation, and survival. Classical NF-κB is a heterodimer composed of the p50 and p65/RelA subunits, which exists in the cytoplasm in an inactive complex. NF-κB is considered a critical link between inflammation and cancer as an aberrant NF-κB activation has been observed in many tumours (Grivennikov et al. 2010).
The primary mechanism for regulating NF-κB is through inhibitory IκB proteins (IκB, inhibitor of NF-kB), and the IκBs kinases (IκK) complex. NF-κB dimers are retained in an inactive form in the cytosol through their interaction with IκB proteins. IκK can phosphorylate IκB, leading to its degradation and consequent NF-κB activation (Oeckinghaus and Ghosh 2009). This permits NF-κB to translocate to the nucleus, activating gene transcription (a wide variety of NF- κB-responsive genes have been identified) (Meng et al. 2002). Aberrant NF-κB activation underlies the development of many cancers. Inflammatory cytokines abundant in cancer, as tumour necrosis factor (TNF)-α and IL-1b, have been shown to activate NF-κB signaling in tumour cells (Chaturvedi et al. 2010).
One well-studied pathway leads to NF-κB activation by the cytokine TNFα and leads to the activation of NF-κB-inducing kinase (NIK), which phosphorylates the IκKs. Recent studies have shown that an alternative pathway, which involves Akt, can phosphorylate IKKs. Signaling pathways involving Akt and NF-κB are known to converge, besides the fact that both are involved in the promotion of cell survival. On the other hand, overexpression of p65/RelA has been shown to stimulate Akt phosphorylation, whereas overexpression of IκB-α reduces Akt phosphorylation. In addition, overexpression of p65 causes an increase in the expression of Akt mRNA and protein (Meng et al. 2002). Akt is involved in the inhibition of apoptosis in certain cancers such as ovarian cancer, prostate cancer, and gastric adenocarcinomas, so the activation of Akt by NF-κB may serve as a strong stimulus in the progression of cancer.
30
INTRODUCTION
1.4.1 ANTISENSE TRANSCRIPT TO PMEPA1: NKILA
Many negative regulators inhibit over-activation of NF-κB signaling and function as tumour suppressors. Among those, there is NKILA, an essential lncRNA, recently discovered, that regulates NF-κB signaling and represses cancer-associated inflammation (B. Liu et al. 2015).
Long non-coding RNAs (lncRNAs) are a large class of non-protein-coding transcripts (greater than 200 bases in length) and involved in numerous physiological and pathological processes (K. C.
Wang and Chang 2012). Recent evidence is growing about lncRNAs regulation on various hallmarks of cancer. However, whether lncRNAs play a role in cancer-associated inflammation is not known yet.
NKILA is induced by NF-κB (NKILA promoter region has a NF-κB binding motif) and it binds to NF-κB/IκB complex inhibiting NF-kB signaling: it masks the phosphorylation sites of IκB and stabilizes the complex. New findings about NKILA have shown that it is divergently transcribed from PMEPA1 and is antisense to some of its transcripts (Figure 5). Whereas the PMEPA1-001 transcript open reading frame and a part of the intergenic promoter region have been described as highly conserved through evolution, it seems to be true to a lesser degree for NKILA equivalent transcripts, so a hypothesis for the function of the seemingly newer NKILA is its possible interference with PMEPA1 expression (Dijkstra and Alexander 2015).
Fig. 5. Schematic view of the PMEPA1 plus NKILA region of human Chr. 20. The figure summarizes several data from the study by Liu et al. for NKILA and its promoter, and PMEPA1 transcripts, which contain or not the NF-κB interfering site, and are overlapping with NKILA sequence.The depicted summary of the PMEAP1 transcripts -001, -002 and -201, is derived from the Ensembl database and agrees with GenBank reports. Exons are indicated by boxes, with protein coding regions in black. The 3’ UTR of PMEPA1 is not drawn in correct proportion to the other exon regions. Arrows indicate the direction of transcription, and genomic regions are measured in basepairs. (Modified from Dijkstra and Alexander 2015).
NKILA A
NKILA AC
31 INTRODUCTION
PMEPA1 function is not totally understood, but it is believed that the encoded transmembrane protein with its cytoplasmic domain could bind SMAD proteins and could affect activation of Akt, as previously described. Signaling pathways involving Akt and NF-κB are known to converge, which could be important in a possible indirect effect of NKILA through PMEPA1 on NF-κB functions (Dijkstra and Alexander 2015).
As NF-κB is the key transcription factor involved in inflammation and is constitutively active in most cancers, many of the signaling pathways implicated in cancer are likely to be networked to the activation of NF-κB. TGF-β activates IKK kinase, which mediates IκB-α phosphorylation. In turn, the activation of IκK by TGF-β is mediated by the TAK1 (transforming growth factor (TGF)-β activating) kinase 1, which belongs to the mitogen-activated protein kinase kinase kinase (MAPKKK) family that can also be regulated by other receptors involved in immune response including the TNFR (Arsura et al. 2003). Furthermore, TAK1 directly phosphorylated the IKK complex in response to TNF-α, IL-1 or LPS treatment, promoting NF-kB activation (Takaesu et al. 2000). As a result of NF-κB activation, IκB-α is increased leading to post-repression of NF-κB and induction of cell death. More research should be done in both NKILA and PMEPA1 regulation of NF-κB signaling pathway.
32
33
O BJECTIVES
34
35
2 OBJECTIVES
The objectives considered for this thesis were based on the first hypothesis for this work that COX2 high expression alters the expression in colon carcinoma cells of some target genes, which might confer a proliferative advantage or migratory/metastatic capacity to these cells.
Once we established PMEPA1 as one of these upregulated genes by COX2 activity, we decided to study the role of PMEPA1 in colorrectal and ovarian cancer. Therefore, the objectives to address were:
1. To study the effects of PMEPA1 overexpression or silencing in colon and ovary cancer cells in vitro and in xenograft mouse models (subcutaneous, peritoneal and orthotopic) of colon and ovary cancer.
2. To analyze the molecular mechanisms responsible of the observed phenotypes, as PMEPA1 implication in TGF-β, WNT/β-catenin and AKT/GSK-3β/β-catenin signaling pathways and if PMEPA1 could interact with components of those pathways.
3. To test PMEPA1 expression in biopsies from patient tumour samples.
4. To analyze PMEPA1 gene transcripts and PMEPA1’s promoter and the possible differential effect of the PMEPA1 alternative transcripts and related non coding RNAs.
36
37
M ATERIALS AND METHODS
38
39
3 MATERIALS AND METHODS
3.1 MATERIALS
3.1.1 Cell lines
The HT29, Caco-2, HCT116, SW620 and SKOV3 cell lines were obtained from the ATCC (LGC Standards, Barcelona, Spain). HT29-lucD6, stably expressing Firefly Luciferase, obtained from Caliper Life Sciences. SKOV3-lucD6 were obtained from Xenogen. SKOV3 were obtained from MD Anderson Cancer Research Center, from Gema Moreno (Madrid, Spain). The SW480 cells were a kind gift from Dr. Alberto Muñoz (Madrid, Spain). The SW620 cells were available in our lab. The Caco2 and HCT116 cells were obtained from the Centro de Investigaciones Biológicas Tissue culture Repository (Madrid, Spain). Cell lines were validated with the StemElite ID system (Promega). Cells were grown in MEM medium (Gibco) containing 5% of fetal bovine serum (FBS), supplemented with sodium pyruvate, glutamine, AANE, antibiotics and G418 (200μg/ml for HT29 or 500μg/ml for SKOV3) –growth medium-, and was changed every 2-3 days.
All cells were maintained in a humidified atmosphere with 5% CO2 at 37ºC.
The cell line HEK293-FT from Invitrogen, from embrionary human kidney was grown in the same conditions as before in DMEM medium with 5% FBS.
3.1.2 Reagents
Name Reference Commercial house
HumanKine Activin A 130-097-608 Miltenyi Biotec
HumanKine BMP4 130-098-786 Miltenyi Biotec
TGFβ1 latent human, recombinant SRP0300 Sigma-Aldrich GW 788388 (inhibitor of TGFβ1 type1
receptor ALK5)
3264 TOCRIS Bioscience
Lipofectamin 2000 11668027 Thermo Fisher
LY2109761 S2704 Selleckchem
TBB (casein kinase II inhibitor I) CAS 17374-26-4 Santa Cruz Biotechnology
Fluprostenol 16768 Cayman Chemical Co
Hygromycin B 10687-010 Invitrogen
40
MATERIALS AND METHODS
Blasticidin Ant-bl-1 InvivoGen
G418 Sulfate Ant-gn-1 InvivoGen
VivoGlo™ Luciferin, In Vivo Grade P1043 Promega
Glutaraldehyde, type II G-6257 Sigma
Crystal Violet C-6158 Sigma
3.1.3 Oligonucleotide primers used for quantitative real time PCR GENE PRIMERS SEQUENCES (5´ 3´)
COX2 F: CGA GGT GTA TGT ATG AGT GTG R: GTG TTT GGA GTG GGT TTC AG
END F: TGA GAG GAA GAA AAA TCA GAA GAA G R: TTT CTC ATG GTC TCC GAC CT
ENG F: GCG GTG GTC AAT ATC CTG TC R: GTT GAG GCA GTG CAC CTT TT ID1 F: CCA GAA CCG CAA GGT GAG
R: GGTCCCTGATGTAGTCGATGA IL2 F: AAG TTT TAC ATG CCC AAG AAG G
R: AAG TGA AAG TTT TTG CTT TGA GCT A INHBA F: TGG CTA TAT GTA GGC AAT GTC AC
R: AGG GCT GCC TCT AGA ACA CA KLF4 F: TGA CTT TGG GGT TCA GGT
R: GTG GAG AAA GAT GGG AGC MPGES 1 F: CTG GTC ATC AAG ATG TAC GTG
R: GGG TAG ATG GTC TCC ATG TC NKILA F: AAC CAA ACC TAC CCA CAA CG
R: ACC ACT AAG TCA ATC CCA GGT G NKILA A F: GCC TTC AGT TCT CCA GCA GC
R: CAT ACC CCT TTT GAA CTC CGC AG NKILA AC F: GCC TTC AGT TCT CCA GCA GC
R: CAC CTT CAC TTC CCA AAT TCC AG NKILA B F: CAG AGA GGG ACG AGA CCT GG
R: CCT ACA TGC CCA GCC CAC ATC
41 MATERIALS AND METHODS
PAI1 F: CCC AGC TCA TCA GCC ACT R: GAG GTC GAC TTC AGT CTC CAG PMEPA001 F: CAG AGC ATG GAG ATC ACG GA
R: CAC CAT CAC CAT CAT CAC CA PMEPA002 F: GAG TTC CCG TCT TTC CTG GT
R: ATG ATC TGA ACA AAC TCC AGC TC
PMEPA1 F: GCC GCC CAC CGC CAG CGA GGT CAT CGG C R: CTA GAG AGG GTG TCC TTT CTG
PMEPA201 F: TTG ACT TCT CCA GAA CAA GCC R: CTA GAC CAG AGC GAA TTC ATC C PTGDS F: CCA ACT TCC AGC AGG ACA A
R: ACA ACG CCG CCT TCT TCT PTGFR F: GGA GAG GCA TGG AGA AGA AAC
R: ATT TAG AAG CCT CAA GGA GAA GG RCAN1 F: GCA TAA GAC TGA GTT TCT GGG
R: AAC TGC TTG TCT GGA TTT GG
SMAD1 F: TGT GTA CTA TAC GTA TGA GCT TTG TGA R: TAA CAT CCT GGC GGT GGT A
SMAD2 F: GCT TCT CTG AAC AAA CCA GGT C R: ATG TGG CAA TCC TTT TCG AT SMAD3 F: CAC CAC GCA GAA CGT CAA
R: GAT GGG ACA CCT GCA ACC SMAD4 F: CCT GTT CAC AAT GAG CTT GC
R: GCA ATG GAA CAC CAA TAC TCA G SMAD5 F: TTG AAG TTA TTG AAG TAC CTG TTG CT
R: TGC TTC TTT CAT TGG GTC AA TGFβ1 F: CAC GTG GAG CTG TAC CAG AA
R: CAG CCG GTT GCT GAG GTA TNFα F: CCC TGG TAT GAG CCC ATC T
R: CTG AGT CGG TCA CCC TTC TC TP53 F: AGG CCT TGG AAC TCA AGG AT
R: CCC TTT TTG GAC TTC AGG TG ZEB 1 F: TGA CTA TCA AAA GGA AGT CAA TGG
R: GTG CAG GAG GGA CCT CTT TA
42
MATERIALS AND METHODS
3.1.4 Antibodies
Antibody name Reference Commercial house Use
AKT1 2938 Cell Signaling Technology WB
p-AKT1 9018 Cell Signaling Technology WB
AKT2 2964 Cell Signaling Technology WB
p-AKT2 8599 Cell Signaling Technology WB
β-catenin 8480 Cell Signaling Technology WB, IF, IP
COX2 clone CX229 160112 Cayman Chemical WB
c-Rel (N) Sc-70 Santa Cruz Biotechnology WB
DAPI 124653 Merck IF
E-Cadherin 3195 Cell Signaling Technology WB, IF, IP
ERK1 Sc-93 Santa Cruz Biotechnology WB
p-ERK1 Sc-7383 Santa Cruz Biotechnology WB
FP Prostanoid Receptor P8622 Sigma-Aldrich WB
GSK-3α/β 5676 Cell Signaling Technology WB
p-GSK-3α/β 9331 Cell Signaling Technology WB
HSP90 Sc-13119 Santa Cruz Biotechnology WB
IκBα 9242 Cell Signaling Technology WB, IP
p-IκBα 2859 Cell Signaling Technology WB
KLF4 ab34814 Abcam WB, IF
LAP (TGFβ) MAB246-100 R&D SYSTEMS Cell culture
mPGES1 10004350 Cayman Chemical WB
N-Cadherin 4933 Cell Signaling Technology WB, IF, IP
NFκB p50 Sc-1190 Santa Cruz Biotechnology WB
NFκB p65 Sc-109 Santa Cruz Biotechnology WB
43 MATERIALS AND METHODS
PAI-1 IM29L Calbiochem WB
PI3Kinase p85 4292 Santa Cruz Biotechnology WB
P21 2947 Cell Signaling Technology WB, IF
SMAD1 6944 Cell Signaling Technology WB, IF, IP
p-SMAD1/5/8 9511 Cell Signaling Technology WB, IF
SMAD2/3 8685 Cell Signaling Technology WB, IF, IP
p-SMAD2/3 8828 Cell Signaling Technology WB, IF
SMAD4 9515 Cell Signaling Technology WB, IF, IP
SMAD5 12534 Cell Signaling Technology WB, IF, IP
Slug 9585 Cell Signaling Technology WB, IF, IP
Snail 3879 Cell Signaling Technology WB, IF, IP
TMEPA1 SAB4502441 Sigma-Aldrich IHC
TMEPA1 WH0056937M1 Sigma-Aldrich WB
TMEPA1 clone 2A12 H00056937-M01 Abnova WB, IF
VEGF Sc-152 Santa Cruz Biotechnology WB
Vimentin 5741 Cell Signaling Technology WB
V5 tag ab27671 Abcam WB, IP
YAP 14074 Cell Signaling Technology WB
3.1.5 Plasmids
3.1.5.1 Reporter plasmids
pALK5/SMAD3-luc (CAGA-luc): consisted of 12 tandem repeats of the upstream SMAD3- binding element from human PAI-1 promoter linked to a viral minimal promoter and to a luciferase gene, and was a generous gift from Aris Moustakas (Biomedical Center, Uppsala, Sweden) (Dennler et al., 1998).
44
MATERIALS AND METHODS
pRCAN1-luc: it was the region from -1664 to +83 of RCAN1 human gene (Cano et al, 2005)
pNFκB-luc: (pNF3ConA-luc) it contains three copies of the consensus sequence κB of the promoter of immunoglobulins κ chain before the conalbumin minimum promotor. Ceded by Dr F. Arenzana-Seisdedos (Institute Pasteur, Paris, France) (Arenzana et al, 1993)
3.1.5.2 Expression plasmids
pLenti CMV/TO Empty Hygro: 8709 bp, carrying Xba-1 and BamH1 restriction sites pLKO.1-puro: 7052 bp, SHC001 from SIGMA, MISSION® pLKO.1-puro Empty Vector Control Plasmid DNA is a lentivirus plasmid vector that contains no shRNA insert.
shPMEPA1: human PMEPA1 MISSION shRNA Bacterial Glycerol Stock (SHCLNG- NM_020182) from SIGMA-Aldrich. Five shPMEPA1 plasmids were ordered:
TRCN0000000331 -shPMEPA1 (E)-: 5’ CCG GGT CCC TAT GAA TTG TAC GTT TCT CGA GAA ACG TAC AAT TCA TAG GGA CTT TTT 3’
TRCN0000000334 -shPMEPA1 (D)-: 5’ CCG GGA GCA AAG AGA AGG ATA AAC ACT CGA GTG TTT ATC CTT CTC TTT GCT CTT TTT 3’
TRCN0000000335 -shPMEPA1 (A)-: 5’ CCG GGA GTT TGT TCA GAT CAT CAT CCT CGA GGA TGA TGA TCT GAA CAA ACT CTT TTT 3’
TRCN0000272440 -shPMEPA1 (C)-: 5’ CCG GGA GTT TGT TCA GAT CAT CAT CCT CGA GGA TGA TGA TCT GAA CAA ACT CTT TTT G 3’
TRCN0000272492 -shPMEPA1 (B)-: 5’ CCG GGT CCC TAT GAA TTG TAC GTT TCT CGA GAA ACG TAC AAT TCA TAG GGA CTT TTT G 3’
pLX304: 9377 bp, Gateway compatible lentiviral vector with a C-terminal V5 tag and CMV promoter for mammalian cell expression; blasticidin resistance in mammalian cells;
ampicillin resistance in bacteria; Gateway cloning. PMEPA1 (Homo sapiens) in pLX304 (Gateway V5-tagged lentiviral expression vector): HsCD00440383 from DNASU (Insert sequence: 757nts)
TCF4-WT: 4797 bp, pDONR223_TCF4_WT, Gateway Donor vector containing TCF4 (Addgene #82166).
TCF4-DN: 9335 bp, TCF4 in pLX303 vector (Addgene #42592).
β-catenin mutant: 6441 bp, pCS2 stabilized mutant Beta Catenin-GFP (Addgene #29684)
45 MATERIALS AND METHODS
3.2 METHODS 3.2.1 Plasmids cloning
Mammalian expression vectors encoding PMEPA1 were generated by PCR amplification of the PMEPA1 open reading frame (transcript 001, human PMEPA1 cDNA, NM_199170
Origene). For PMEPA1- pLentiCMV/TO vector, the primers
5'ATGGATCCATGCACCGCTTGATGGGG 3' and 5'TACCTCTAGACTAGAGAGGGTGTCCTTTC 3', carrying Xba-1 and BamH1 restriction sites, were used for plasmid amplification in the PCR. The PCR product was digested with Xba-l and BamH-1 overnight at 37ºC. The digested DNA was run on an agarose gel and was purified with GeneClean III KIT (Promega). The plasmid was subcloned into the pLentiCMV/TO Hygro vector at 14ºC overnight, and verified by DNA sequencing. The ligation reaction was transformed into competent cells and afterwards it was grown in cultive plates with LB medium with the resistant antibiotic for each plasmid (Ampicillin or Kanamycin) overnight at 37ºC. Colonies grew and individual bacterial colonies were picked to check them for successful ligation. Afterwards, more colonies were picked to be grown in tubes with 5 ml LB medium with correspondent antibiotic, overnight at 37ºC. DNA was obtained with Wizard® Plus SV Minipreps DNA Purification System.
To obtain the three different PMEPA1 transcripts, the primers 5’CACC ATG CAC CGC TTG ATG GGG GTC 3’ and 5’GAG AGG GTG TCC TTT CTG TTT ATC CTT CTC 3’ were used for PMEPA1- 001 plasmid (3400 pb approximately) amplification in the PCR; 5’CACC ATG GCG GAG CTG GAG TTT GTT CAG 3’ and 5’GAG AGG GTG TCC TTT CTG TTT ATC CTT CTC 3’ were used for PMEPA1- 201 plasmid (3000 pb approximately) amplification in the PCR and 5’CACC ATG ATG GTG ATG GTG GTG GTG ATC AC 3’ and 5’GAG AGG GTG TCC TTT CTG TTT ATC CTT CTC 3’ were used for PMEPA1-007 plasmid (3200 pb approximately) amplification in the PCR. Process was performed as previously described, while these plasmids were subcloned into pENTR/D-TOPO vector, 2580 pb (Addgene) and then into pLX304 vector (V5-tagged plasmid) with GATEWAY RNA Clonase II Enzyme Mix (Invitrogen), using as a template the pENTR/DTOPO (2580 bp) plasmid. DNA was obtained also as above described.
3.2.2 Transient cell transfection
12 μg of plasmids (PMEPA1 or EV pLenti Hygro) were used to transfect semi-confluent HT29-LucD3 and SKOV3-LucD3 cells in 100mm Petri dishes with Lipofectamine 2000 reagent (Invitrogen). Cells were recollected after 72 hours for a particular experiment. The COX2- overexpressing cell lines and the empty vector controls (EV) were generated by transfection with
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MATERIALS AND METHODS
the pBabe-puro vector carrying or not the human COX2 gene, using Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer.
3.2.3 Lentiviral cDNA transfection
All lentiviral plasmids and the packing plasmids were co-transfected into HEK293FT cells using Lipofectamine 2000 according to the instructions of manufacturer. Lentiviruses were collected at 72 h after transfection and used to transduce PC-3 cells in a 24-well plate. Twenty-four hours after transduction, Blasticidin (15 µg/ml) (for the pLenti system) and Puromycin (2 µg/ml) (for the pLKO.1 puro shRNA system) were added to select drug-resistant cell populations.
Lentiviral plasmids co-transfection (pLenti PMEPA1 or empty vector –EV-, pLenti Hygro, pCMV 8.2 and pMDG) and lentivirus collection was done as described before. The pLenti Hygro Empty vector was used as a negative control. All lentiviral plasmids and the packing plasmids were co-transfected into HEK293FT cells using Lipo-fectamine 2000. Lentiviruses were collected at 48 h and 72 h after transfection with 8µg/ml of Polybrene and used to transduce HT29 and SKOV3 (luciferase expressing and not) cells in a 24-well plate. At the third day after transfection, Hygromycin (200μg/ml) was added to select drug-resistant cells until cell death of those not infected; subsequently, cells were treated with Hygromycin and G418 (200μg/ml) for maintenance of the expression of luciferase. In the case of COX2 overexpressing cell lines, cells were selected for one week with 2µg/ml Puromycin. Then several independent mass cultures were obtained.
3.2.4 Cell proliferation assays
Cell proliferation assays were performed plating 20000 cells in p35 wells by cell counting using the Neubauer haematocytometer. Cells were plated in 3 or 4 different wells and each day of the experiement cells from a different well was counted.
Cell proliferation was also quantified by Crystal Violet staining. Cells were washed two times with PBS before they were fixed in 5% Glutaraldehyde in PBS for 20 minutes. Fix solution is removed and cells were stained with 0,5% Crystal Violet in water and 50% methanol for 20 minutes. Crystal Violet solution should be washed by immersion in water tap 3 times and cells are left until total dry. Crystal Violet should be diluted with Acetic Acid 10% during 5 minutes at room temperature, measuring immediately D.O. at 570nm.
