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31/10/2018

  hPG 80 , circulating progastrin ,

a new target to fight cancer  

Note to readers:

Numerous works carried out jointly by the scientific and medical communities around the world have demonstrated or aim to demonstrate the multiple possibilities that hPG80 (circulating progastrin) offers in helping cancer detection and diagnosis, in accompaniment of therapeutic monitoring, in the monitoring of recurrences or in the treatment of cancer itself, alone or in combination with other therapeutic means.

Today, the scientific knowledge gathered on progastrin and the mechanisms of its interaction with cancer are sufficiently demonstrated and solid to be made available to practitioners so that they can in turn define the best clinical methods of use.

and integrating these new means in their fight to serve patient health.

We called on Dominique Joubert Floch, PhD in biology, and Alexandre Prieur, PhD in oncology, to carry out an objective and complete scientific review of the link between progastrin and cancer, which synthesizes and puts into perspective all of the work made, discoveries made and evidence gathered by numerous laboratories since 1990.

Dominique Joubert Floch and Alexandre Prieur are affiliated with the company ECS Progastrin Lab.

hPH80, THE CIRCULATING PROGASTRIN,

AND ITS LINKS TO CANCER

FROM THE DISCOVERY OF PROGASTRIN TO THE TUMOR REVERSION.

This review addresses a major issue: the role of progastrin in cancer.

However, in order to get into this subject with a comprehensive understanding, we will first provide some general information, starting with a question:

What is a tumor?

A tumor is a heterogeneous collection of cells of which 1 to 5% have a phenotype of cancer stem cells. These cells ensure the survival of the tumor and must therefore be the subject of specific therapies (Kaur et al., 2018). They are able to migrate and invade surrounding tissue and form distant metastases; they are capable of generating the cells which form the mass of the tumor: the progenitor cells which themselves can enter into a differentiation program, often incomplete (Figure 1).

Figure 1.jpg

Figure 1 : Complexité et hétérogénéité d'une tumeur adaptée de (Hanahan et Weinberg, 2011)

Tumor cells, like normal cells, do not have a stable phenotype (da Silva-Diz et al., 2018). This means that a progenitor cell, for example, could revert to a stem cell if the tumor has an increased need for stem cells (Figure 2).

It is therefore crucial for the eradication of tumors to target both cancer stem cells and other cells. Today, the vast majority of therapies target proliferating cells, that is, progenitor cells. This is the case with chemotherapy or therapies that target the mechanisms that ensure cell proliferation (Figure 3).

In addition, the growth of a tumor requires the formation of new vessels (neo-angiogenesis), in order to supply the tumor cells with the growth factors and oxygen necessary for their survival and proliferation. Cancer stem cells can survive in an environment unfavorable to other cell types such as hypoxia or the absence of growth factors. They can also survive chemotherapy treatments using intracellular mechanisms capable of excluding chemotherapy molecules from the cell, which makes them resistant to these treatments (Batlle and Clevers, 2017).

Figure 2.jpg

Figure 2 : plasticité des cellules cancéreuses adaptée de (da Silva-Diz et al., 2018)

Figure 3.jpg

Figure 3 : cible moléculaire pour des thérapies ciblées adaptées de (Li et Li, 2014).

The link between progastrin and cancer has been known for more than 30 years. Progastrin is involved in most of the properties of cancer cells that ensure the existence of the tumor: proliferation, survival of cancer stem cells in normoxia and hypoxia, cell migration and invasion, angiogenesis, intracellular mechanisms responsible for the different properties of tumor cells ( figure 4).

Figure 4.jpg

Figure 4 : La progastrine, pierre angulaire des voies oncogènes

This document is an objective and comprehensive review of the link established by many laboratories between progastrin and cancer. We will analyze these links as we go along, starting with the following question: what is progastrin?

WHAT IS PROGASTRIN?

In 1905, John Sydney Edkins showed the existence of a hormone responsible for the secretion of gastric acid. This hormone was called gastric secretin, or gastrin.

But it was not until 1979 [partial sequence of mRNA: (Noyes et al., 1979)], then in 1987 and 1988 [precursor of human gastrin: (Desmond et al., 1987; Dockray, 1988) ] that progastrin was identified as the precursor to gastrin. Its sequence has been elucidated as well as the sequence of its mRNA.

Progastrin is a peptide of 80 amino acids, processed in the endoplasmic reticulum, gastrin being the final active product of the maturation of progastrin.

Figure 5 shows how successive cleavages of progastrin lead to the end product, gastrin (Copps et al., 2009). Sulfation and phosphorylation both play a role in the maturation process: they both increase the processing of progastrin, while phosphorylation can also affect the conversion of gastrin intermediates to extended glycine (G34-Gly and G17- Gly) in mature gastrins (Bishop et al., 1998).

Figure 5.jpg

Figure 5 : Transformation de la progastrine humaine adaptée de (Copps et al., 2009).

WHERE IS PROGASTRIN EXPRESSED IN PHYSIOLOGICAL CONDITIONS?

Progastrin is mainly expressed in the stomach, where gastrin is secreted by the G cells of the antrum. The main function of gastrin is to regulate acid secretion.

The other maturation products, in particular G34-Gly, G17-Gly and CTFP, have been assigned various functions, in particular the CTFP (C Terminus Flanking Peptide) described in order to be able to induce or inhibit apoptosis depending on the tissue or cell type. concerned (Marshall et al, 2013; Patel et al, 2010; Smith et al, 2006)

Progastrin has also been shown to be expressed in other tissue extracts (cerebellum, pituitary gland, pancreas, testes), but to a lesser extent than in the stomach, and the role that progastrin / gastrin can play in these organs are often not clearly understood (Bardram, 1990; Rehfeld, 1986; Rehfeld, 1991; Schalling et al., 1990). In the testis, for example, it is the carboxyamide forms of gastrins that are present in sperm. The normal pancreas also expresses the mRNA of gastrin, and it is assumed that the gastrinomas expressing progastrin originate from the pancreatic cells secreting progastrin.

THE LINK BETWEEN PROGASTRIN AND CANCER: HOW HAS IT BEEN SHOWN?

Bardram was the first to hypothesize that "a low degree of transformation of progastrin could serve as a predictor of a malignant clinical course at an early stage of the disease" (Bardram, 1990). He reached this conclusion after assessing the presence of progastrin and its products in the serum of patients with Zollinger-Ellison (an endocrine disorder characterized by an overproduction of gastrin due to a tumor (more often malignant than benign) or endocrine hyperplasia, most often located in the pancreas). He noted that the total progastrin product better reflects the tumor synthesis of gastrin than the conventional measures of alpha-amidated gastrin.

After this observation, many publications have described the presence of the expression of progastrin in cancers, starting with the first evidence of the use of a human gastrinoma where the petides derived from progastrin were purified and characterized (Huebner et al., 1991).

However, progastrin has been shown to be poorly treated in cancer cells due to the absence or ineffectiveness of the processing enzymes. And this has been clearly demonstrated in colorectal cancer (Ciccotosto, 1995; Finley et al., 1993; Imdahl et al., 1995; Kochman et al., 1992; Nemeth et al., 1993; Singh, 1994; Van Solinge and al., 1993b). Indeed, Kochman has shown that in the colon tissue, progastrin is more than 700 times more abundant than amidated gastrin. In contrast, gastrin amidated in the human antrum is the predominant form of gastrin by a factor of 10. This has been confirmed by Nemeth et al using a different approach. Separation on Sephadex G50 revealed that most colorectal carcinomas contain peptides derived from the precursor of gastrin, progastrin, but most of these tumors do not transform progastrin into biologically active products. Immunostaining also showed that in a series of 23 adenocarcinomas, more than 50% of the tumor cells were stained for gastrin and progastrin. Singh et al. then demonstrated that progastrin was not fully treated in human colon cancer cell lines and, most importantly, that it was secreted from these cultured cells in vitro, paving the way for analysis of an autocrine / paracrine functional function of progastrin in tumor cells (Singh, 1994; Van Solinge et al., 1993b) and Figure 6). Colorectal cancers are not the only type of cancer to express progastrin. Ovarian cancers do too, although progastrin concentrations are much lower than those of amidated gastrin (van Solinge et al., 1993a) as are liver tumors which express precursor forms of gastrin, in particular progastrin unlike normal liver (Caplin et al., 1999).

Pancreatic tumors also express the gastrin gene, with 91% of tumors with the untreated progastrin product (caplin, 2002).

Figure 06.jpg

Figure 6 : La progastrine est exprimée dans le cancer colorectal humain. L'expression de la progastrine a été analysée dans les tumeurs primaires de 12 patients et dans les métastases présentes chez ces mêmes patients. Les résultats sont présentés sous forme de multiplication par deux de l'expression dans la métastase par rapport à la tumeur primaire appariée.

Thus, progastrin is expressed in different types of tumors and is secreted in vitro from cancer cells. Therefore, the next question is: Can we detect progastrin in the blood of cancer patients?

PROGASTRIN IS PRESENT IN THE PLASMA OF PATIENTS WITH COLORECTAL CANCER

The evidence that progastrin could be detected and quantified in the blood of patients with colorectal cancer was demonstrated by Siddheshwar et al as early as 2000 ((Siddheshwar et al., 2000) and Figure 7).

These authors provided definite data on the increase in plasma levels of progastrin and unamidated gastrin in patients with colorectal cancer compared to a control series. They also studied a series of patients with adenomatous polyps and also observed an increase in progastrin, even if this increase was not statistically significant, unlike that observed by Prieur et al in 2017 (Prieur et al., 2017 ). In this later work, progastrin was tested with a very sensitive Elisa sandwich test. This technical approach made it possible to demonstrate the increase in the level of blood progastrin in 67% of patients with adenomatous polyps.

It is interesting to note that Siddheshwar et al. also showed that fasting total plasma levels of gastrin in the blood of colorectal cancer patients were also higher, regardless of positive or negative Helicobacter pylori status. This is due to untreated gastrins since the levels of amid gastrins have remained unchanged.

Thus, progastrin is present in the tumor and in the blood of patients with colorectal cancer. But does tumor progastrin represent all of the blood progastrin?

This was demonstrated by Konturek et al in 2002 (Konturek, 2002). These authors measured progastrin in the blood of colorectal cancer patients before and after surgery. Rates increased as expected in colorectal cancer patients compared to controls before surgery and returned to normal values after surgery.

All the arguments were therefore gathered for the scientific community to begin to analyze the function or functions of progastrin on tumor cells. The reasoning was there and, as you will see below, the results have indeed demonstrated the major role that progastrin exerts on the tumor, providing the ground for considering today progastrin as a new target for the fight against Cancer.

Figure 7.jpg

Figure 7 : La progastrine est présente dans le plasma des patients atteints de cancer colorectal.  

La progastrine a été dosée dans le plasma de patients atteints de cancer colorectal et dans une série de témoins à l'aide d'un essai radioimmunologique. Les patients atteints du cancer colorectal présentaient une concentration de progastrine significativement plus élevée que les témoins (adapté de (Siddheshwar et al., 2000)).   

PROGASTRIN AND ITS FUNCTIONS ON CANCER CELLS

In order to understand the importance of the role of progastrin in the regulation of cancer cells, it is crucial to understand how a tumor is initiated and how it evolves. And the best model for doing this is the colorectal cancer model.

Colon tumorigenesis begins about 30 years before colon cancer becomes symptomatic. The first event leading to colon cancer is the constitutive activation of the wnt / ß-catenin oncogenic pathway induced by the mutation of the APC (most common) or ß-catenin gene. It has been shown that the introduction of these mutations into normal stem cells of the intestine is indeed sufficient to initiate tumorigenesis (Huels and Sansom, 2015). These mutations induce the formation of an adenoma with preneoplastic characteristics, then the evolution towards an adenocarcinoma. Then, other mutations occur, leading to activation of other oncogenic signaling pathways (Figure 8). To reach this stage of tumor development, the cells had to proliferate, to become independent of neighboring cells (what is called contact inhibition). Next, tumor cells must acquire an EMT (Epithelial-Mesenchymal Transition) phenotype in order to invade adjacent normal tissue and possibly form distant metastases. In addition, for the tumor to grow, new blood vessels must be generated, called neoangiogenesis, and the cells must escape immune monitoring in order to escape recognition of T cells.

All of these characteristics are controlled by intracellular mechanisms, among which certain signaling pathways are of paramount importance.

Figure 08.jpg

Figure 8 : Représentation schématique de la tumorigenèse intestinale, montrant l'accumulation de mutations et l'activation des voies de signalisation (adapté du projet Syscol).

We will now provide the evidence from the literature that demonstrates that progastrin is involved in the majority of the mechanisms that tumor cells use to survive and grow.

Progastrin and proliferation of tumor cells

As early as 1996, expression of the gastrin gene was shown to be necessary for the tumorigenicity of cancer cells in the human colon (Singh et al., 1996). Singh and his coauthors studied the functional role of the gastrin gene by examining the effect of expression of gastrin antisense RNA (AS) on the growth and tumorigenicity of colon cancer cells (resulting in inhibition progastrin production). The proliferative and tumorigenic potential of AS clones of gastrin-expressing cell lines was significantly reduced compared to that of control clones. Based on these observations, the authors predict that the growth of a significant percentage of colorectal cancers may critically depend on the expression of the genetic products for gastrin.

Among the progastrin maturation products, gastrin extended to glycine can also play a trophic role on tumorigenesis. Indeed, Hollande et al. in 1997 (Hollande et al., 1997) showed that gastrin extended to glycine acts as a trophic factor in untransformed cells, then Stephan et al. in 1999 (Stepan et al., 1999) showed that gastrin extended to glycine stimulates the growth of HEK cells and human colon cancer cells in vitro.

Progastrin is a promoter of intestinal tumorigenesis.

To demonstrate the role of progastrin in intestinal tumorigenesis in vivo, mouse models were generated, either mice deficient in the gastrin gene, or mice overexpressing the gastrin gene.

In 1997, Koh et al. generated mice deficient in gastrin (Koh et al., 1997). They observed that the colon was histologically normal, which indicates that progastrin does not play a major role in the physiology of the colon.

Then, transgenic mice overexpressing human progastrin without specific tissue targeting (hGAS) or in the form of transgenic insulin gastrin (INS-GAS) were produced (Wang et al., 1996). The pancreatic islets of INS-GAS mice were able to produce carboxyamide G-17, which resulted in a double elevation of serum amid gastrin, marked thickening of the oxyntic mucosa and an increased proliferation index of the gastric body. In contrast, the hGAS adult mouse livers expressed an abundance of human gastrin and human progastrin mRNA, but were unable to process this peptide to its mature amidated form, which resulted in markedly serum progastrin levels high and levels of normal amid gastrin. These mice had an increased proliferation index in the colon, suggesting that incompletely processed gastrin precursors may contribute to the proliferation of the lining of the colon in vivo. Overexpression of gastrin extended to glycine in transgenic mice has also resulted in increased colonic proliferation (Koh et al., 1997).

In these genetically modified mice, colonic proliferation increased but did not result in the formation of a tumor. Clearly, progastrin was not a tumor initiator. Evidence that progastrin could be a tumor promoter was then obtained in mice predisposed to tumor development.

Two experimental arrangements were used:

1 / mice overexpressing progastrin are treated with azoxymethane (AOM), a chemical carcinogen (Cobb et al., 2004; singh, 2000; Singh et al., 2000) resulting in a significant increase in tumor formation.

2 / mice carrying a mutation in the APC gene (APCmin / +) are crossed with mice deficient in gastrin (Koh et al., 2000). In the APCmin / + mouse, an allele of the APC gene undergoes a mutation leading to its inactivation. Thus, each time a cell loses the second allele of APC, the Wnt / ß-catenin pathway is activated constitutively, initiating intestinal tumorigenesis, with first spontaneous adenomas then adenocarcinomas. In APCmin / + mice deficient in gastrin, there was a marked decrease in the number of polyps and a considerably reduced rate of polyp proliferation.

Pannequin et al. in 2007 (Pannequin et al., 2007) and Prieur et al. in 2017 (Prieur et al., 2017) used another mouse model carrying a different mutation in the APC gene called APC∆ / +. These mice, like APCmin / +, spontaneously develop adenomas and adenocarcinomas, but with more of these tumors in the colon. In both studies, progastrin was altered by treating mice with siRNA (Pannequin et al., 2007) or with a neutralizing anti-progatsrin antibody (Prieur et al., 2017). It is interesting to note that, as for APCmin / + mice, in all cases, the inhibition or neutralization of progastrin leads to a reduction in the number of tumors (Figure 9).

Figure 9.jpg

Progastrin is essential for the survival of cancer stem cells.

 

These cancer stem cells represent a small proportion of the tumor, they are thought to represent between 1 and 5% of the tumor. But they are especially important for the survival of the tumor because they play the role of "reactor". Without them, the tumor does not survive. they have the capacity to self-renew and generate all the other types of cells present in the tumor by asymmetric division, starting with the progenitors which have a strong propensity to proliferate (figure 10).

Cancer stem cells do not proliferate at a high rate. They therefore escape treatments that target proliferating cells, such as chemotherapy. They can migrate and invade the surrounding tissues and are therefore at the origin of distant metastases (Batlle and Clevers, 2017).

While targeting cancer stem cells is crucial, this cannot be done without also targeting other cells.

Indeed, the phenotypes are "plastic" and a progenitor cell can revert to a phenotype of cancer stem cells if there is an increased need for cancer stem cells (Figure 2).

It was therefore important to understand whether the in vivo effect of progastrin in intestinal tumorigenesis could involve regulation of cancer stem cells.

Such a role has been suggested by the observation that progastrin is expressed in CD133-positive colorectal cancer cells, which express some of the phenotypic characteristics of cancer stem cells (Ferrand et al., 2009).

However, it was Giraud and his co-authors who really demonstrated the major role that progastrin plays in cancer stem cells (Giraud et al., 2016).

They first showed that the expression of progastrin, both at the level of the mRNA and at the level of the protein, was greatly increased in colorectal cancer cells cultured under conditions where the cancer stem cells are enriched (conditions non-compliant, spherical test).

Progastrin then proved to be mandatory for the formation of spheres that need a cancer stem cell to start growing.

This indicated that progastrin could regulate the frequency of cancer stem cells, which was demonstrated in vitro and in vivo thereafter (Figure 11).

Figure 9 : Nombre de tumeurs dans le tractus intestinal des souris APC∆/+ traitées avec un anticorps témoin ou anti-progastrine (adapté de (Prieur et al., 2017))    

 

Ces résultats mettent en évidence le rôle de la progastrine comme promoteur tumoral. Comme c'est essentiellement la progastrine qui est sécrétée par les cellules tumorales et non les produits maturés, la progastrine peut donc représenter un événement précoce dans la tumorogenèse colorectale et peut contribuer significativement à la progression tumorale.   

Figure 10.jpg

Figure 10 :Caractéristiques des hiérarchies de cellules souches cancéreuses (en rouge sur le schéma) adaptées de (Batlle et Clevers, 2017).

Figure 11.jpg

Figure 11 : L'appauvrissement en progastrine nuit à la survie et à l'auto-renouvellement in vitro des CSC adaptés de (Giraud et al., 2016).

Later, Prieur et al. also demonstrated that migration and invasion, two characteristics of cancer stem cells, are both greatly affected in vitro and in vivo (Figure 12).

Figure 12.jpg

Figure 12: anti-progastrin antibody inhibits migration (A) and invasion (B) of colorectal cancer cells adapted from (Prieur et al., 2017).

In addition, it is the secreted progastrin which plays the role of a survival factor for cancer stem cells. In fact, when a neutralizing antibody is added to the culture or when mice grafted with human colorectal cancer cells are treated in vivo with such an antibody, the frequency of cancer stem cells is also reduced ((Prieur et al., 2017). ) and Figure 13).

These two articles demonstrate that progastrin is a factor in the survival of colorectal cancer stem cells. Progastrin could thus help target the heart of the tumor, and therefore the tumor itself.

Figure 13.png

Figure 13 : l'anticorps anti-progastrine diminue la fréquence du SCC des cellules cancéreuses colorectales (adapté de (Prieur et al., 2017)).

BY WHAT MECHANISMS DOES PROGASTRIN CONTROL THE FORMATION OF TUMORS?

Progastrin decreases apoptosis

As we have already mentioned, progastrin is able to stimulate the proliferation of tumor cells. Wu et al have shown that it is also capable of reducing apoptosis (Wu et al., 2003). This has been demonstrated in intestinal epithelial cells sensitive to gastrin cultured in the presence of progatsrin. A significant loss in the activation of caspases 9 and 3, leading to a significant loss in DNA fragmentation during PG processing of cells, has been observed.

Thus, the effect of progastrin on cell survival results from both an increase in proliferation and a decrease in apoptosis.

 

Progastrin regulates adhesions and tight junctions.

 

For a cell to proliferate and migrate, it must become independent of neighboring cells. The integrity of cell-cell contacts is therefore essential for preventing the formation of metastases, which first required cell migration. Hollande et al. in 2003 (Hollande et al., 2003) demonstrated the major role that progastrin plays on cell-cell junctions, whether they are adherent or tight. In human colorectal carcinoma cells DLD-1 secreting progastrin, the expression of an antisense gastrin constructs the restored membrane localization of the protein constituting these junctions (zonula occludens-1 (ZO-1), occludin, β- catenin and E-cadherin). This effect was reflected both by an increase in the activity of the tyrosine kinase Src and by the induction of a spatial delocalization of the protein kinase Cα (Hollande et al., 2003).

 

Progastrin is a pro-angiogenic factor.

 

When a tumor grows, it needs additional oxygen and nutrients from new blood vessels. The production of new blood vessels is called neoangiogenesis. Recently, in 2015, progastrin was found to be a pro-angiogenic factor, which means that it induces the formation of blood vessels (Najib et al., 2014). Progastrin has stimulated proliferation and migration of endothelial cells and increased the ability of endothelial cells to form capillary-like structures in vitro. In vivo, when progastrin production was blocked by shRNA in xenografted cells in nude mice, the neovascularization of the tumor decreased. These observations, combined with a mechanistic understanding of vascular endothelium-cadherin, p125-FAK and paxillin, provided the evidence necessary to demonstrate the role of progastrin as a pro angiogenic factor.

Progastrin and hypoxia

A tumor is not a homogeneous set of cells, especially because there are areas, especially in the middle of the tumor, that are less vascular than the rest of the tumor. In these particular areas, hypoxic conditions are therefore a constraint for the cells present.

Cancer stem cells have learned to resist hypoxic conditions. They can survive in this harsh environment when other types of cells dye.

The first evidence of a link between progastrin and hypoxia was provided by the work of Laval et al. who have shown that in vivo, the overexpression of progastrin provides a physiological advantage to mice under hypoxic conditions (Najib et al., 2014). Later, in 2017, Prieur et al showed that in vitro, the expression of progastrin is stimulated under hypoxic conditions, which corresponds to the fact that cancer stem cells express higher levels of progastrin than other cells. tumor (Prieur et al, 2017). Progastrin could therefore help cancer stem cells to survive under hypoxic conditions.

Thus, progastrin, through a variety of mechanisms which are all crucial for the growth and survival of tumors, can be considered a major tumor promoter. Its main function is to help cancer stem cells survive and spread to form metastases, which probably explains why progastrin can also be considered as a potential predictor of liver metastases in colorectal cancer (Westwood et al. , 2017).

Figure 14.png

The questions are now:

  • How can progastrin perform these functions?

  • What is the progastrin receptor capable of transducing its signal?

  • What are the intracellular mechanisms involved?

  • Does progastrin have a direct link with oncogenes?

Figure 14 : Chronologie des principales dates qui décrivent le lien entre la progastrine et le cancer, de la découverte de la gastrine à la démonstration que la progastrine est une nouvelle cible dans la lutte contre le cancer.

WHAT IS THE RECEPTOR FOR PROGASTRIN?

Despite continued efforts by the scientific community to identify and characterize the progastrin receptor, the progastrin receptor is not yet clearly identified. We will now review all the candidates and try to understand why it is unlikely that they are the true progastrin receptor.

What is certain is that the receiver exists.

High affinity binding sites were first described in IEC cells using recombinant iodinated human progastrin. The affinity was of the order of 0.5-1 nM, which was compatible with a receptor. When the binding of biotinylated progastrin to cells was evaluated by flow cytometry, a strong and specific binding of progastrin to certain cell lines (IEC-6, IEC-18, HT-29, COLO320) was also detected (Dubeykovskiy et al., 2008). Binding specificity was confirmed by competition with unlabeled cold PG, but not with glycine prolonged gastrin or amid-17 gastrin. Binding was not influenced by the presence of the classical CCK-2 receptor.

It is clear from these two studies that there exists a site or a receptor for binding to progastrin distinct from the binding of gastrin amidate 17 and of prolonged gastrin to glycine 17. The sequence of progastrin interacting with this receptor probably found in amino acid residues 26 of progastrin at the COOH-terminal end of progastrin which have been shown to be sufficient for progastrin function (Ottewell et al., 2005), but the identity of this putative receptor remains an open question.

One of the candidates is Annexin A2, identified as capable of binding progastrin and the peptides derived in 2006 by Singh et al (Singh et al., 2006). Annexin A2 is a partial mediator of the effect of progastrin / gaztrine. In particular, Appendix A2 mediates in the up regulation of NF-κB, β-catenin and stem cells in response to progastrin in mice and HEK-293 cells (Sakar et al, 2011). In addition, Annexin A2 may be implicated in the endocytosis of clathrin-mediated progastrin (Sarkar et al., 2011). However, the affinity of progastrin for annexin A2 is not that expected for a specific receptor. And, although Annexin A2 plays a role in the functions of progastrin, it is not that of a receptor.

Another recently suggested candidate is the G protein-coupled receptor 56 (GPCR56), expressed on both colon and cancer stem cells (Jin et al., 2017). Indeed, while the recombinant human progastrin favored the growth and the survival of colonic organoids of wild type in vitro, the colic organoids cultivated on GPR56 - / - mice were resistant to progastrin. However, although progastrin has been shown to bind cells expressing GPCR56, the authors have not provided evidence of direct binding to GPCR56 itself. GPCR56 is a good candidate, but evidence that it is THE progastrin receptor is still lacking.

The progastrin receptor is capable of activating a number of signaling pathways, directly or indirectly, which is rather unusual for a receptor. This could indicate a peculiarity of this receptor, which is why it is difficult to identify.

The unidentified progastrin receptor transduces the progastrin signal via various intracellular intermediates known to be involved in tumorigenesis.

PROGASTRIN AND ONCOGENIC SIGNALING PATHWAYS

The first demonstration of the link between progastrin and the oncogenic pathway has been described for K-ras. Indeed, colon cancer cell lines and tissues with K-ras mutations all had significantly higher levels of gastrin mRNA than those of the wild-type K-ras (Nakata et al., 1998). The effects of K-ras on gastrin expression occurred by activation of the Raf-MEK-ERK signal transduction pathway, the final step being activation at the level of the gastrin promoter.

The ras oncogene oncogene p60-Src, the first oncogene identified, is activated in cancer cells of the colon by increasing the quantities of progastrin (Brown, 2003), which means that the production of progastrin which also occurs early during tumorigenesis (Pannequin et al., 2007) could play a role in this activation, also known as an early event in colonic tumorigenesis (Cartwright et al., 1990; Iravani et al., 1998). PI3K / Akt, involved in proliferation in particular, is also activated by progastrin (Ferrand et al., 2005; Pannequin et al., 2007).

Another major signaling messenger regulated by progastrin is NF-kappaB. Its involvement in the mechanisms responsible for the anti-apoptotic effect of progastrin has been demonstrated in pancreatic cancer cells in vitro (Rengifo-Cam et al., 2007) and in vivo in mice overexpressing progastrin (Umar et al. , 2008).

Upward regulation of kinases activated by Janus2, STAT3 and kinases regulated by the extracellular signal has also been observed in the colonic mucosa of hGAS (Ferrand et al., 2005).

Of all these regulations, however, the most important is the link between progastrin and the Wnt pathway, which provides the essential understanding for progastrin to be considered a target in the fight against cancer today.

PROGASTRIN AND THE WNT PATH

The Wnt pathway has been known for its involvement in tumorigenesis for many years, in particular for the survival of cancer stem cells (Bhavanasi and Klein, 2016; Nusse and Clevers, 2017).

In colorectal cancer, the Wnt pathway is constitutively activated in 80 to 90% of tumors, with a somatic mutation in the APC gene in the majority of cases.

There are many genes whose expressions are activated by the Wnt oncogenic pathway. The gene coding for progastrin is one of them. Indeed, Koh and his colleagues have shown that the gastrin gene is a target downstream of the ß-catenin / TCF-4 signaling pathway and that the co-transfection of a ß expression construct -catenin with constitutive activity causes a triple increase in the activity of the gastrin promoter (Koh et al., 2000).

It is a foundational work for understanding the link between progastrin and cancer due to the many cellular functions involving the Wnt pathway in a cancer cell, starting with its importance for the survival of cancer stem cells.

Since the K-Ras and Wnt pathways both induce expression of the progastrin gene, it was then hypothesized that there could be cooperation between the two pathways in regulating the expression of progastrin.

This is indeed what Chakladar and his co-authors observed (Chakladar et al., 2005). They found strong synergistic stimulation (25 to 40 times) of the gastrin promoter by the combination of the oncogenic ß-catenin and the overexpression of K-ras.

Activation of the gastrin promoter could be further enhanced or suppressed by co-expression of wild type SMAD4 or a dominant negative mutant of SMAD4, respectively, and repealed by the PI3K inhibitor. Thus, the constitutive activation of the Wnt pathway, considered to be at the origin of colon tumorigenesis, and the oncogenic K-ras, present in 50% of human colorectal tumors, synergistically stimulate the production of progastrin, a promoter of tumorigenesis.

HOWEVER, AND OF CAPITAL IMPORTANCE, IT WAS FOLLOWED THAT THE DESTRUCTION OF PROGASTRIN MAY INACTIVATE THE WNT PATH AND REVERSE TUMORIGENESIS.

How were these conclusions established?

As described above, progastrin is a target gene for ß-catenin-Tcf4 transcription factors. But does progastrin exert a feedback mechanism on this path and, if so, is it positive or negative feedback?

The strategy to answer this question was simple: decrease the production of progastrin via siRNA, then measure the transcriptional activity of ß-catenin-Tcf4 using a luciferase test. Colorectal DLD-1 cancer cells have been transfected, and the results have shown that when progastrin production is impaired, the transcriptional activity of ß-catenin-Tcf4 is deeply inhibited (Pannequin et al., 2007 ).

Thus, progastrin has a positive feedback on the activity of ß-catenin-Tcf4. The mechanism for this feedback has been clarified. It involves PI3K, ILK and ICAT. ICAT is an endogenous inhibitor of the ß-catenin-Tcf4 interaction. When expressed, TCAI binds to ß-catenin, preventing it from being associated with Tcf4. The two transcription factors delocalize in the cytoplasm, causing de facto inactivation of the pathway (Pannequin et al., 2007).

The Wnt pathway, constitutively activated in colorectal cancer cells due to a somatic mutation, can be inactivated, which should not have been possible at the time when this work was carried out. The consequences of this inactivation have been analyzed at different levels, including that of cell differentiation. Pannequin et al. have shown that tumor cells that do not express progastrin return to a normal state.

This is due to the fact that when the Wnt pathway is inactivated, a gene called serrate-1 is downregulated, which induces inactivation of the notch pathway which plays a major role in the acquisition of a differentiated phenotype ( Pannequin et al., 2009).

Cancer cells begin to express the Muc2 gene, proof of their reacquired functional differentiation.

The consequence of the inactivation of the Wnt and Notch pathways by inhibition of the production of progastrin has also been observed in a mouse model which recapitulates intestinal tumorigenesis, the mouse model APC + / +. These mice were treated either with siRNA (Pannequin et al., 2007) or with anti-progastrin antibodies (Prieur et al., 2017). In both cases, the number of tumors that develop spontaneously in the intestine has decreased, which indicates that tumorigenesis due to APC is indeed dependent on progastrin.

The fact that inhibition of the Wnt pathway induces a "reversal" of tumorigenesis was demonstrated by Dow et al in 2015 (Dow et al., 2015). The authors of this work crossed mice carrying mutated K-Ras (KRASG12D) and P53 fl / fl with mice having an inducible APC of shRNA RNA. When APC was not expressed, Wnt activity was high, tumors developed. When APC was expressed, Wnt activity was low, tumors disappeared, despite the presence of KRASG12D and P53 fl / fl.

It demonstrated that targeting the Wnt pathway is sufficient to reverse tumorigenesis.

P53 is a tumor suppressor gene thought to be essential for tumor progression.

In 2012, the mutation in the P53 gene was shown to increase progastrin-dependent colonic proliferation and the formation of colon cancer in mice (Ramanathan et al., 2012).

Progastrin is therefore a factor used by the cancer cell to survive and evolve over time. The more the tumor progresses, the higher its dependence on progastrin. Targeting progastrin could therefore represent an effective tool in the fight against cancer.

PROGASTRIN AS A TARGET IN THE FIGHT AGAINST CANCER

Until now, progastrin has not been considered a target for cancer.

We have now provided data here that demonstrate that this should change for two main reasons: progastrin is found in the plasma of cancer patients and neutralization of progastrin induces tumor reversion .

Progastrin is found in the plasma of cancer patients. Progastrin is detected from the preneoplastic stages, such as adenomatous polyps. But since progastrin is produced by tumor cells in the primary tumor and in metastases, it is reasonable to suggest that progastrin can be used for monitoring patients. Circulating gastrin concentrations have been observed to increase in patients at risk of developing colorectal carcinoma (Paterson et al., 2014). It has also been observed that the expression of progastrin in hyperplastic polyps is observed in the very rare cases that have progressed to cancer (Do et al., 2012; Do and Seva, 2013). In addition, progastrin may also be a biomarker of liver metastases in colorectal cancer (Westwood et al., 2017).

Regarding progastrin as a therapeutic tool, the fact that cancer stem cells need progastrin to survive is fundamental since there is currently no drug capable of targeting cancer stem cells (Prieur et al., 2017) .

Targeting progastrin also sensitizes tumor cells to radiation therapy, which could help radiation therapy to be more effective (Kowalski-Chauvel et al., 2017).

In addition, chemotherapy induces a spectacular increase in progastrin in colorectal cancer cells, in vitro and in vivo (Prieur et al., 2017). This corresponds to the fact that cancer stem cells escape chemotherapy, probably partly thanks to the production of progastrin which helps them to survive.

These observations indicate that a combination of chemotherapy (or any other antiproliferative drug) with anti-progastrin antibodies could be very effective in targeting both proliferating and cancer stem cells.

All the necessary data have been generated and published to support the rationale for progastrin as a new target in the fight against cancer.

The scientific community has recognized the role of progastrin in the development of cancer. Now is the time for oncologists to take a closer look at this data and work with scientists to develop the tools that will work for patients in the fight against cancer.

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