THE CANCER STEM CELLS AND LUNG CANCER

POPESCU IULIAN PhD,MD, Clinical Department of Radio-Biology at the Fundeni Clinical Institute in Bucharest

e-mail popdociul @yahoo.com

Dr. ALINA HALPERN PhD, SF.ŞTEFAN Hospital  Bucharest

It is well recognized that lung localization of the cancer is one with the weakest therapeutic results, with all scientific conquests as concerns the therapies obtained from 1950 until today. Surviving more than 5 years is below 15%, compared to results from other localizations (99% in prostate cancer, 86% in breast cancer, 66% for colon cancer, 30% in ENT sphere cancer). In lung cancer encouraging results have been obtained in the possibility of increasing the survival time without relapse (disease free survival), but not also concerning the overall survival. The main task in the treatment of lung cancer remains the prolongation of overall survival and

further increasing the relapse-free survival time.

But there is still hope with a possible degree of achievement: the targeted treatment of cancer stem cells. The data to be presented represent the scientific basis of a future effective treatment. However the doctor cannot disregard the treatment of growing the body’s defense capacity. This important thing remains on a secondary, but not neglectible place. The true therapy will be the targeted treatment of the cancer stem cells together whithe the treatment for increasing the fighting capacity of the body.

CHAPTER I STEM CELL (STRAIN CELL)

Their isolation was possible by their ability to eliminate the Hoechst 33342 colouring matter, a fluorescent dye, which together with bromo-5-deoxiuridine colour the nuclei in order to distinguish the compact chromatin out of the apoptotic nuclei, can identify the cells that replicate and can sort cells depending on their DNA content. (2)

Stem cells have an ability to restore a population similar to the original one, have a capacity to initiate tumor compared to non-stem cells, have a high potential of agressivity. They still exist in a small but persistent number.

Most of them are in the G0 stadium of the cell cycle. Have a resistance to polychemotherapy (since they have a low level of mRNA MCM-1 which highlights the complex DNA replication (2). The main characteristic would be their ability of self-recovery and this can be seen in the tissues with high turnover.

Self-renewing means that normal stem cell population keep constant their number of symmetric and asymmetric combinations of the cell division (3, 4, 5).

The growth mechanism of normal stem cells population, but also in tumor development would be achieved by increasing the symmetric cell division (two daughters resembling mother cell) as opposed to asymmetric division (a stem cell daughter, the other daughter is non-stem cell). Stem cells have a capacity of self-renewing, but are relatively quiet and their cell cycle is more lenghtened in time than the cell cycle of proliferative non-stem cells (6.3). This would due to the stopping in G 0 phase of the cell cycle.

Another property of stem cells is the differentiation potential in several cell lines, because only progenitor cells similar to stem cells (stem-like) can give rise to different cell types.    

The stem cells of the normal tissue are also dependent on the interactions with adjacent stromal cells that comprise a specialized area (niche). This is necessary for maintaining the stem cell identity and their capacity for self-renewing. Here is regulated the amount of stem cell, their proliferation, stem cell resistance to apoptosis. The niche has a complex structure and is composed of:    

a) various stromal cells (immune cells, mesenchymal cells),    

b) vascular network    

c) soluble factors    

d) components of the extra cellular matrix.

EMBRYONIC MARKERS OF STEM CELLS (7)

1)OCT-4(octamer-4). Is a transcription factor. It is expressed in embryonic stem cells, totipotent germ cells. It is encoded by the POU-5A (8) gene. It is a regulator of normal stem cell pluripotency and is the most recognized marker of all-potent embryonic stem cells. It requires a critical level of OCT4 for the support and renewal of stem cell and their pluripotency. Its over or subregulation leads to developing divergent programs. For instance, the ectopic expression of OCT-4 blocks the progenitor cell differentiation and causes dysplazia in the epithelial tissue (9) OCT-4 is involved in the self-renewal of the embryonic stem cell. It is a marker of undifferentiated cells, while the differentiated cells have a low expression. OCT-4 supports the self-renewal ability of adult stem cells in epithelia, liver, bone marrow (10).

2) ssEA(stage specific Embryonic antigen). It has been identified by 3 monoclonal antibodies.

3) CD34, CD33, ABCG2, Sca-1. They are markers of the hematopoetic stem cells.

4) STRO-1 marker of the mesenchymal (stromal) stem cells.

5) NESTIM: PSA-ENCAM (polysalicilic acid neural cell adhesion molecule)

P75- neurotrophic R (NTR). Marker of the neural stem cells.

The pluripotent stem cells express 4 transcription factors essential:

OCT-4, SOX2, KLF-4, NANOG.

The development of various organs during embryogenesis includes specific signaling pathways: Sone hedgehog (SHH), Notch, Pten, Bm-1, WNT, p53.

The disturbance of these pathways - in the course of tumor development - is supposed to lead to disordered self-renewal and contribute to neoplastic proliferation (11).

While stem cells are well-defined functionally, there have also been used some markers to identify stem cells. However, these markers data, have a relative value in characterizing the stem cells. A great success would be finding a negative marker for the stem cell isolation (7).

.

Ways of controlling the stem cells regulation:

Bm-1(polyclonal group transcriptional repressor Bm-1). It was found in lymphoma (13) as an activated oncogene and later was noticed that it specifically regulates the haematopoetic stem cells (14).

The NOTCH pathway. It has a role in the control of haematopoetic stem cells, of normal stem cell and in the control of mammary stem cells (15, 16)

SONE HEDGEHOG and WNT, which are involved in the cell growth process, are also involved in the stem cells regulation (17).

Lung stem cell markers

1)ABCG2(BCRP). The ABCG2 gene encodes the protein ATB-finding cassette sub-family G member2. Is a cyto-protector of normal and malignant stem cells (18 )

2)ALDEHY DEHYDROGENASE. They are a group of enzymes catalyzing the oxidation (dehydrogenation) of aldehydes. It is associated with the survival in lung cancer.

3)Ep-CAM(epithelial cell adhesion molecule). Is a protein encoded by the gene with the same name. It is expressed in undifferentiated pluripotent stem cells.

4)SCF R/c-KIT. The stem cell factor receptor (which is a gene product of the proto-oncogene c / KIT) and its ligands play a role in gametogenesis in hematopoesis. Is a member of a sub-family of receptor tyrosine kinase (RTK). It is expressed in hematopoietic progenitor cells, germ cells, neurons, glial cells, kidneys, lung, intestine.

CHAPTER II

CANCER STEM CELL (CSC)

HYSTORY. The CSC study began in 1990. In 1994, Lapidot T et al separated for the first time the CSC of the acute myeloid leukemia in humans (26,19). In the following 5 years has been developed the research on CSC in solid tumors: breast (26,20), prostate (26-8), brain (26-21), pancreas (26-23)) colon cancer (26 24) and lung cancer (25,26). DEFINITION. The organ regeneration in mammals requires a potential common background of stem cells or progenitor cells (27). Tumors are a heterogeneous cell population, organized ierarchically with a normal stem cell compartment with self-renewal capabilities and multiple differentiation (28).

It is difficult to give a clear definition. It is a cell in a tumor that has the ability of self-recovery (self-renewal), and can reproduce cancer cells heterogeneous which are contained in a tumor, through stem cells mechanisms (29). The cells hold the main properties of stem cells, being rare in frequency, entering in an infrequent cell cycle, constituting a population that is intrinsically resistant to current therapies, these therapies being able to kill only cells in cycle (29).

It is believed that these cells are a category of primitive, undifferentiated cells having the self-renewal ability and differentiation in various cancer cell types (30). Plays a high role in metastasis and they are resistant to cytotoxic agents (31). Within the tumor they are a distinct population, being the cause of relapses.

THE CSC ORIGIN. It is difficult to define their origin, but researches are ongoing.  

The first theory holds that they arise by the normal stem cells mutation and their progenitors. Normal stem cells can become CSC by the disorder of the pathways proliferation, differentiation, or by inducing oncoprotein activities.  

The 2nd theory holds the possibility of their apapearance from the mutation of the cell population from the niche.  

The 3rd theory holds that it would due to the dedifferentiation process.  

The 4th theory supports the concept of tumor hierarchy. The tumor would be a formation as an hierarchic structure, with mutant cells, which may vary depending on the specific tissue environment. To a certain specific appear the CSC cells, the other adapting over time.

CSC CHARACTERISTICS.

The CSC characteristics are those of stem-like cells. Thus we have:

-Self-renewal ability,

- Increased resistance to toxic, chemical aggressions. They are resistant to radiotherapy and chemotherapy

-Capacity of differentiation. The proliferation generates more CSC, but also all kinds of differentiated cells that make up the tumor.

-The CSC form new tumors if they are xeno-transplanted in immunodeficient animals

-They are pluripotent.

-Have strong tumorigenic capacity.

-Non-CSC-cells have low potential for tumor initiation.

MECHANISM OF SELF- RECOVERY (SELF- RENEWAL)

The main property of stem cells is the ability to self-recover. This is observed in cells with an increased turnover (33) and in the tissue repair after injuries. Self-recovery implies that normal stem cell population keeps its number of cells through the symmetric and asymmetric combination of the cell division (34,35). The growth mechanism of the stem cell population in tissue repair, as well as in tumor growth would be achieved by increasing the symmetry of cell division in contrast with the asymmetrical division. The cells with a high expression of OCT-4 have the self-recovery ability and represent a potential proliferative basin of generating cancer cells (36). Stem cells have the ability to self-recover, but they are relatively quiet cells, their cell cycle being longer in time, unlike the cell cycle of the non-stem proliferative cells (37). This would be due to stopping the cell cycle in G0 phase.

MECHANISM OF RESISTANCE TO VARIOUS AGGRESSIONS

The current treatments (radio and chemotherapy) have a curative potential only if they sufficiently inactivate the cancer stem cells. The CSC inactivation after radiotherapy treatment brings a control of the CSC, whereas the persistence of CSC survival leads to relapse (38, 39,40). But CSC mediates their intrinsic radio resistance and through the high cell number before treatment. The intrinsic resistance would be due to expression of glutathione-s-transferase and synthase tymidilat (41).

Another resistance factor is the cells ability to repair and recover among the radiotherapy fractions. During treatment the increased resistance would be due to more factors:

-tumour hypoxia

-environmental factors (high expression of lactate (39)

-increased fraction of positive (+) CD133 cells as a marker of CSC (42), which makes it resistant to treatment.

-by fractioning the treatment in case of radiotherapy (43). The increase of CSC cells is highlighted by Ki67 index (proliferation index).

-increasing the CSC resistance to treatment compared to non-CCS cells is due to the increased potential for defense against ROS.

-high expression of lactate in the respective cells, leading to cellular radio-resistance.

-CSC mediate radio-resistance by tumor-specific factors:

a)number of cells,

b)capacity to recover and

c) degree of reoxygenation.

-Hypoxia reversely correlates to radio-sensitivity (44, 45, 46). Today hypoxia is visualized and quantified using positron PET tomography (45,47). The hypoxia modulation raises the local control value (48).

The outcome evaluation following radiotherapy is performed using CCS. One of the techniques is the markers highlighting, which allow thes visualization and helps us to determine the local control after radiotherapy.     

For the first time it was highlighted a surface marker related to CSC, which may specify the control degree after radiotherapy (49, 50). This marker is CD44mARN and CD44 histochemical. It has been proved a correlation between local control after radiotherapy and CD44 expression. The CD44 expression is correlated with the cell number and stem cells density, being able to indicate the degree of healing (39,40, 51,52). CD44 expression helps us to individualize the treatment (50)).

It has been found a correlation among the CD133 pozitive cells (surface antigen) and resistence-related proteins. Since the CD133 expression is linked to the resistant phenotype. The detection of CD133 positive cells may be useful to clarify the chemotherapy efficiency. The CD133 positive cells, but not CD133 negative cells, express the high levels of markers with multiple resistances to drug such as cis-platinum, doxorubicin, paclitaxel and radiotherapy as, for example, ABCG2.     

Also there is an obvious connexion between the increased expression of OCT-4 and CD133 positive cells.    

The decrease in OCT-4 expression within the CD133 positive cells may inhibit their ability of tumor invasion and colony formation, while increasing the activity of Caspase 3 and PAPR (poly (ADP-ribosil polymerase). A large number of CD133 positive cells is related to drug resistance (36).

DIFFERENTIATION. Another characteristic of stem cells is the differentiation potential for several cell lines. It has been suggested that stem cells could be the origin of these cell lines, because only stem cell-like progenitor cells can give rise to various cell types.     

It is known that tumors are heterogeneous populations (32, 54), and tumors would act as aberrantly developed organs (32, 55).     

The organ development during embryogenesis includes specific development pathways: SHH, Notch, PTEN, Bm-11 WNT, p53. The disorder of these pathways during tumor development - it is believed - that leads to the disorder of self-recovery and contributes to neoplastic proliferation (32,11).

The diagnostic value of cancer stem cell markers for being able to isolating them.

It is difficult to define a cell with stem capabilities only based on surface markers, because none of surface markers is expressed exclusively by cells with stem attributes: CD44, Sca-1, Thy-1. Many cells show these markers without having the capabilities of stem cell types.     

-The markers used for isolating the stem cells capabilities of an organ are not useful to identify the cells with stem capacities of another organ. These markers may be or not useful to identifying the cells with stem capacities.     

-A method to distinguish the cells present in the SP (Side Population) population is the method HOECHST (33324) DYNE EFFLUX.

The distinct cells identification by this method does not mean that we have been able to identify cells with stem characteristics since this attribute is not universal.     

-Other method for identifying cells with stem capacities is the incorporation of 5-bromodeoxyuridine. This is based on the fact that stem cells have long periods of the cell cycle and then can keep longer the incorporantion of 5-bromodeoxyuridine. But - unfortunately - not all stem cells are marked with 5-bromodeoxyuridine, neither all marked stem cells are cells with stem properties.    

-Another method would be the highlighting of genes and signaling pathways, which have a function of regenerating the tissue - in vivo - so they could be isolated: Bm-1, SHH, NOTCH, WNT-betaCatenin. Neither this method is specific as these genes and signaling pathways frequently operate in other cells.    

-Only the operational methods are valid in order to prove that the cells have the ability to regenerate tissues in vivo in order to isolate them. (29)

TUMOR INITIATION AND ROLE OF CSC

Tumorigenesis in humans involves two central theories:    

The first theory: tumor formation and its growth are the increased proliferation of the cells in cancerous tumor compared with the normal tissue.The theory was based on:     

- a series of mutations that lead to the activation or overexpression of genes forwarding the proliferation (oncogenes).      

- supressing the tumor suppressor genes.      

- elusion of apoptosis.

All this leads to tumor growth and then to the process of metastasis. But therapies based on these data have failed to cure the disease. (32)      

A second theory: the tumorigenic cell mutations are few and they last a long time into the tissues. To become tumorigenic, this rare population, with mutated cells, gets abilities of self-recovery, clonal development and to acquire new mutations. It is considered that these non-common cells are tissue stem cells (or other cells derived from them) that obtain self-recovery properties. When these cells are mutated they become cancer stem cells. Reality indicates that these mechanisms are interrelated. It is proven that a cell mechanism explains how genetic changes lead to tissue changes in all phases of tumorigenesis. The disease initiation involves the development of a population of type CSC (32). Both stem cells and cancer stem cells represent only 1% of a certain tissue, but significantly in advanced cancers. The fact that in advanced tumors the number of cells increases exponentially (10⁸ up to 10¹³) it is logically deduced that the stem cells subpopulation will grow exponentially. This occurs at all stages of tumorigenesis (32). By definition the CSCs will renew.

It is suggested that CSCs would initiate and lead to tumor growth in contrast with other non tumorigenic cells.

Thus : First theory: Cancer arises through disturbing the self-recovery process of tissue stem cells or progenitor cells.            

Second theory: the CSCs have tumorigenic capacity and lead to tumor growth. Both stem cells and CSCs are resistant to current treatments, leading to therapeutic failure. Current treatments only target differentiated tumor cells but not target the relatively dormant CSCs. 

The only effective treatment is to target these CSCs (32)

The third theory holds it would be due to the differentiation process.

A fourth theory is the concept of tumor hierarchy.The tumor would be a hierarchic structure formation with mutant cells, which may vary depending on the specific of tissue environment. To a certain specific, cells appear as CSC, the other cells adapting along the way (29).

THE NICHE

The CSCs seem to benefit from the surrounding tissue (niche) being ableto keep the ability of self recovery, but can give rise to other offspring, more differentiated, while they remain undifferentiated (28). The niche defends the cells from geno-toxic insults and contributes to enhancing the resistance. The surrounding microtissue plays a role in the spread during the primary tumor progression focusing on distinguishing the non-CSC cells. Through niche is inserted the epithelial mesenchymal transition (EMT), which in the end is involved in the formation of premetastatic niche, with a role in metastasis formation (28). There are connections among EMT, CSCs and tumor cell invasion (28).

Metastases are usually in the bones, liver, brain, lungs. This has led to the "Seed and Soil" assumption. According to this, the local microtissue seems to be more responsive to the receipt of disseminated tumor cells compared to other organs. Metastases do not occur randomly and the disseminated cells go where they can find a hospitable environment so as to start a new tumor. It seems that the primary tumor is also involved in adapting these pre-metastatic niches necessary to tumor cell that is to arrive, secreting systemic factors and directing cells derived from bone marrow or macrophages to those receiving areas. (56,57).

Hematopoietic cells derived from bone marrow, which are also VEGFR-1 positive and lead to localizing in the pre-metastatic areas, forming nests before reaching the tumor cells (56). Eradicating these cells from bone marrow can prevent the formation of pre-metastatic nests, blocking the metastasis, myofibroblasts, existence of fibronectin deposits in these areas. Most of them link to VLA-4, a fibronectin receptor, which is the expression of hematopoietic progenitor cells. All facilitate the accumulation of these cells.

In conclusion the CSC niche is important in various stages of tumorigenic cascade. In primary tumors, the CSC niche is an important regulator of stem cell quality. This is supported by the fact that the loss of these niches leads to the CSC disappearance. The niche (besides the fact that it maintains the CSC deposits and primary tumors), plays a role in the non-tumorigenic cell transformation into CSC through processes related to epithelial mesenchymal transition, that lead to invasion and metastasis.

Several working assumptions for the future:

It is not known yet whether the pre-metastatic niche is able to install a CSC phenotype in the differentiated cells and also we do not know the role of the surrounding microtissue - in tumor growth, progression and metastasis.

The role of epithelial mesenchymal transition in forwarding the CSC phenotype

The CSC surrounding microtissue has a role in the preservation and protection of compartment. Thus the myofibroblasts are able to dedifferentiate the tumor cells, transforming the non-tumorigenic tumor cells, from well differentiated cells in poorly differentiated cells (by reactivating WNT) with CSC characteristics, having a high tumorigenic potential (58).

Thus, the CSC niche induces a CSC phenotype that leads to dedifferentiation of differentiated tumor cells.     

Tumor cells possess a degree of plasticity that allows them to change their phenotype and to acquire various functions and properties under the influence of the environment.        The epithelial mesenchymal transition (EMT) and mesenchymal epithelial transition (MET) are processes that reflect the tumor cell plasticity. These processes are characteristic of embryonic period and carcinogenesis (59).       

The tumor cells suffering of EMT gain aspects of CSC (60). EMT leads to dissemination of tumor cells with self-recovery, proliferation capabilities leading to the dissemination and development of the metastasis.         

The fact that EMT promotes the CSC prototype makes this process to be controlled and regulated.

In promoting the CSC phenotype also intervene:

1) HYPOXIA (derived from the surrounding microtissue) regulates the cell plasticity. Hypoxia forwards the ability of self-recovery and stem-cell phenotype within the framework of non-stem population, increasing the capacity to form spheres that also regulate important factors of the stem cells (OCT-4, NANONG, cMyc. (59) Another mechanism of hypoxia leading to the ability to invade is the WNT-way action, which, in turn, induces the transcription factor SNAIL (61). The SNAIL protein expression was detected in the tumor-stroma interface (62).

2) In addition to hypoxia, other factors intervene in the surrounding microtissue, such as TGF-beta, which stimulates the proliferation and expansion of the CSCs, which furnther leads to metastasis (63,64,65).

3) The ability of differentiated cells to acquire stem cell properties is also made ​​by using myofibroblaştilor. These cells are important within the TEM-CSC network.

CHAPTER III

CANCER STEM CELLS (CSC) and LUNG CANCER

Organ regeneration in mammals requires a potential common background of stem cells or their progenies, but their role in lung regeneration is not well known (66).The current assumption - very attractive - suggests that lung cancer originates in adult stem cells, through a process of malignant transformation in various human organs.

These transformed cells are known as cancer stem cells (31).         

The normal lung tissue is made ​​up of a variety of cell types (68):

-basal cells that secrete tracheal and bronchial mucus,

- CLARA cells from bronchioles,

- type I and type II alveolar epithelial cells,

It is an extremely complex organ, being renewed conditionally, which anatomically and functionally contains distinct stem populations that are located in distinct anatomical areas.

These mature cells derive - through differentiation - from lung progenitor cells. These, in turn, are formed from undifferentiated and multipotent stem cells (69). Like other stem cells, lung stem cells maintain the normal tissue structure and repair the injuries (70)).

The basal cells, CLARA cells and alveolar-capillary type II pneumocytes are candidate cells to be stem cells or their progenitors. They can repair injuries and contribute to the normal tissue restoration.

Kim et al have isolated a stem cells population residing in a niche in the broncho-alveolar duct junction. (BASC) (71,72). They have been identified and characterized as CD34 pozitive cells, Sca-1 positive and CD45PECAM negative and expressing CCSP (Clara cell secretion protein) and pro-surfactant protein C, which are markers for CLARA cells and for alveolar epithelial type II cells.

It has been proven that BASC have a self-renewal capacitaty and are multipotent.

(72). BASC proved to have a resistance in case of bronhiolar and alveolar injuries and proliferated during the cell epithelial renewal - in vivo. BASQUE have expanded - in culture - as a response to the Kras oncogene, and the precursors of lung tumors - in vivo. These data support the assumption that BASC are a stem population that maintains the CLARA brochiolar cells and alveolar -epithelial cells in the distal lung area and that transformed can be the precursor cells of lung adenocarcinoma. They can become cancer cells by origin after induction by the Kras gene in codon 12 (71).

BASC Cells contribute only between 0-25% to supplementing the epithelial and alveolar cells of type I and II. Thus the regeneration requires an essential contribution of alveolar-epithelial type II cells. There is a linear hierarchy for achieving the pulmonary recovery: BASC-AEC II-AEC I (66).

BASC and AEC II have proliferative features, while AEC I remain differentiated cells. The AEC II have an important role in the expansion and healing of alveolar compartment. Thus AEC II cells (or an AEC II population) have a function of reparative progenitor cells. The BASC cells do not derive from type II alveolar epithelial cells, but it is not yet known which are the precursors of the BASC cell.

The BASC cells are the first cells that increase their number, being a subpopulation highly proliferative and continue to proliferate for a long time, playing a role in the lungs recovery.

Type II alveolar-epithelial cells are progenitors of the alveolar-epithelial cells of type I. In the tissue recovery process, the alveolar-epithelial type II cells are the prevalent factor. The quantitative role of the participation of BASC cells and type II epithelial cells socket is under clarification.

There are necessary further investigations for clarifying whether the return to the original number after the end of BASC cell proliferation is achieved through differentiation or cell death.     

It is not yet specified if the BASC derived from CLARA cells or epithelial cells or alveolar type II, or a precursor yet unidentified.      

The cells with stem cell properties of the CLARA cells contribute more to restoring the bronchioles’ local growth than in the distal expansion of the alveolar - epithelial type I cells.(66)

Links with lung adenocarcinoma

-The cancer cells with phenotype of brochiolar stem cells are detectable in pulmonary adenocarcinoma (67).  

- The OCT-4 gene was detected in several solid tumors (73). The stem cell with BASC properties express the presence of OCT-4 protein in lung adenocarcinoma.   

- The OCT-4 gene expression regulates the self-recovery of these cells, which induces a severe prognosis (67).        

- The type II alveolar epithelial cells, CLARA cells and those of terminal brochioles and BASC cells are at the origin of preneoplastic lesion of lung adenocarcinoma, adenomatous hyperplasia-induced by the Kras gene (67,74).

-the Kras expression facilitates the transformation of broncho-alveolar stem cells which continue to give rise to adenocarcinoma after the naftalen treatment. This suggests that BASC cells may be a target in the tumor turning to lung cancer (68.72). But unfortunately, it is an overlapping between the stem-cell antigen in mice with the human one.   

The role of CD133 positive cancer cells

It has been succeeded to isolate and characterize the population of CD133 positive cancer cells (75) in non-small cell lung cancer. They are able to form spheres and act as cells that initiate tumor in non-small cell cancer. For their isolation have been used:

a) Flow cytometry of positive cell for the markers CD24, CD29, CD31, CD34, CD44, CD133, CD326.

b) HOECHST 33342 dyne exclusive test has been used to identify the population characterized by the presence of stem cell (Side Population).

In lungs only indirect evidence has been brought about the existence of CSC in the lung tissue. Stem-like cells have been identified in the lung of mice of the population able to induce malign transformation (72). But human lung tumors show a phenotypic heterogeneity, suggesting that it could originate from a multipotent cell (71).

Like other stem cells, the lung stem cells maintain the normal tissue structure and repair the injuries (70).

Lung stem cells express typical antigens of undifferentiated cells such as (CD34, BCRP-1 (breast cancer resistance protein-1) (76,77). Authors have identified a rare population of CD133 positive cells as a tumorigenic cell population of lung cancer. A small number of CD133 positive lung cancer cells were able to reproduce tumors in the immuno-compromised mice while CD133 negative cell population did not show this. An unlimited number of CD133 positive cells was obtained in both sub-types of lung cancer, using selective culture conditions, that allowed their expansion as tumor areas. The lung cancer areas have proven that there is a differentiated cell phenotype showing the expression of CD133 positive and the lack of cell markers specific to cell lines. These data suggest that LC (lung cancer) may be initiated and propagated by stem-like cells not differentiated.     

Cancer stem cells (CSC)-CD133 positive have the ability to generate lung cancer cells differentiated under adequate culture conditions. This is evidenced by the fact that it acquires markers specific to cell lines while losing the CD133 expression.

These differentiated cells are phenotypically similar, with most of such tumor cells. This indicates the existence of a hierarchical model for furthering lung cancer tissue. This is based on the fact that we have a generation of many progenitor cells, from a small number of undifferentiated cells which are self-recovering.     

Similar to tumorigenic cells from other tumors, lung cancer CD133 positive cells are capable of self-recovery, extensive proliferation. They are able to grow as undifferentiated cells over a year, keeping the ability to reproduce the original tumor after the transplantation on immuno-compromised animals.     

The number of lung cancer areas endowed with the capacity of self-recovery is between 5% to 30%, measured by clonogenic methods.

As the expression of CD133 positive is quite homogeneous, it is likely that CD133 positive lung cancer cells comprise two stem-like cell populations with similar phenotype, but with different potential:

- a stem-like cell population capable to recover and

- the second non-tumorigenic population of progenitor cells with limited potential for proliferation.

The tumorigenesis potential of lung cancer areas is shown by the fact that a small number of CD133 positive cancer cells were able to reproduce- by transplanting - original tumor, both morphologically and histochemically (68).

The authors have identified in mice two CLARA cell subtypes, which would be the population to repair the lung and are resident in the neuro-epithelial corpora and and broncho-alveolar duct junction.

Neuro-epithelial corpora are islands containing amines and peptides, which are released under conditions of hypoxia.

The CLARA cells in the surviving neuro-epithelial corpus population may supply both neuroendocrine and epithelial cells.

The second type of surviving CLARA cells is localized la broncho-alveolar duct junction and seem to play a role in the regeneration of the epithelial components of the broncho-alveolar structure.(78).

These broncho-alveolar cells are capable to recover and show all regional stem cells features in the distal lung (79).

The CD133 positive cells from human cancer and broncho-alveolar stem cells in mice expression share the same extension of the stem cell markers expression: CD34, BCRP-1, OCT 4 and have the CD45antigens absent (79).    

The authors observed that in mice the rare CD133 positive cells increase considerably after the injury with naphtalen according to the possibility that CD133 positive cells in mice-be expressed in broncho-alveolar stem cells.     

In contrast, the broncho-alveolar stem cells and type-1I alveolar-epithelial cells have a variable expression in lung cancer areas, reflecting the CSC phenotypic diversity in humans.

In the experiment, the lung cancer areas containing a significant percentage of stem-like cells are capable to renew. But this is also linked to the respective cell line. This is seen from the strict similarity between their offspring and tumor subtype.     

In lung cancer is unlikely that everything derive from a single primitive multipotent stem cell, but rather the various subtypes of lung cancer are maintained by aberrant immature cells engaged in various cell lines. As proof is the fact that besides the common expression of CD133 positive, the lung cells show a changeability of the proliferation rate, differentiation potential, the number of clonogenic cells which develops in long-term cultures.     

On the role of CD133 positive expression there are also recent works (80), which bring more punctual data on the role of CD133 expression, but reverse and even contradictory to the above shown :

-it is assumed that tumors contain a small CSC population (numerically lower) which initiate the tumor growth and promotes the dispersion. The resistance to treatments allows the tumors to escape the conventional therapy.

-CD133 is one CSC marker in some cancers,including the lung.

-CD87 is related to stem-like characteristics in small cell lung cancer.

- the CD133 positive and CD87 negative subpopulations, as well as the CD133 negative şi CD87 positive populations have shown a high resistance to etoposid and paclitaxel treatments and an ability to repopulate only the CD133 negative and CD87 negative subpopulation (!)

-the CD133 positive and CD87 negative populations contain more cells in G0 stage than the CD133 negative and CD87 negative cells.

-by contrast, the CD133 and CD87 negative population have shown a high tumorigenic potential.

To conclude - after the authors (80) CD133 and CD87 are not suitable markers for CSC (!). But they can predict resistance to chemotherapy.

Other authors (81) believe that CD133 is a marker for small cell lung cancer (on H446 cell lines). CD133 positive cells have stem characteristics and tumorigenic capacity in small cell lung cancer.

Circulating tumor cells

Recently, the circulating tumor cells (CTC) have acquired an interest representing biomarkers with possible prognosis and being able to predict the metastasis.

There have been found methods for their identification:

-immuno-magnetic technology consisting of nano-magnetic particles wrapped with anti-EpCAM antibodies. So far it is the best technique for the CTC detection approved by the FDA. This technique was used before chemotherapy in breast cancers, colon, prostate. Their listing was used for their prognostic value and for monitoring the response to chemotherapy (82).     

It can also be used in early stages prognosis to identify patients requiring adjuvant therapy for pursuing the occurring of relapses.

This technique can also be used in genetic and molecular characterization of the CTC:

-increased number of pre-operative CTC is correlated with a severe prognosis (83)

CTC-basic number and its modification after one chemotherapy cycle is an independent prognostic factor in small cell lung cancer (87).

-the postoperative CTC increasing number correlates with high risk of relapse (84).

-there is a correlation between the CTC number and extent of disease. (85)

- in small cell lung cancer, in 86% of cases with high CTC number have severe prognosis (86).

-in the case of non-small cell lung cancer, if the number of cells is larger than the number 5 is a bad prognostic sign and vice versa, the CTC number decreases after a cycle of chemotherapy is correlated with the increase of free disease survival (disease free survival) and overall survival, in comparison with the increase of CTC number (88).      Regarding the metastatic potential it has been calculated in animals that approximately 2.5% of CTC give rise to micro-metastases and only 0,01% give macro-metastases (89).

In relation to metastasis developments, in addition to CSC profile also matters the modification of epithelial phenotype (the epithelial mesenchymal transition (EMT).

The transition from epithelial phenotype to mesenchymal phenotype occurs through the progressive loss of epithelial specific markers (cyto-keratin and EpCAM) and the increased expression of Vimentin as a sign of mesenchymal phenotype (82).

The CTC from Lung Cancer is correlated with the prognosis both in early cases and in the late ones.

In conclusion , the CTC of lung cancers helps us to stratificate the patients in different therapeutic strategies.     

Determining the CTC number is a non-invasive virtual biopsy.     

The therapeutic anti-CTC treatment prevents metastasis.     

The future therapeutic targets will be stem cells and also epithelial mesenchymal transition.

Few opinions on the current treatments limits

In practice it has been proved how difficult it is to assess the healing degree of the lung cancer due to large tumor heterogenity and tumor microenvironment (49) Both stem cells and cancer stem cells are resistant to current treatments, which leads to treatment failure . The current treatments, radio and chemotherapy have only a limited role as they target only the differentiated tumor cells and do not target the cancer stem cells. (90,91,92,93). Current therapies do not target either the signaling pathways that regulate self-restoration, as they are mutated or epigenetically deregulated. These are: SHH, NOTCH, PTEN, Bm-1, WNT, p53(94).

The role of CD44. There are more variations of CD44v. These isoforms of CD44 are involved in the motility and proliferation cellular functions (95). Targeting these variants seems to be a promising treatment strategy. In the experiment it was observed that the combination of cytotoxic chemotherapy with directed anti-CD44 antibody plus fractionated radiotherapy have improved the local control (96). The first step was taken towards the clinical application of surface markers related to stem cells as predictors of the tumor radio-sensitivity .      

Another therapy problem was observed in cases of non-small cell lung cancer, accompanied by EGFR mutations and being treated with tyrosine-kinase inhibitors. Following treatment has been generated a population of stem properties.These cancer cells with stem properties are - intrinsically – cells resistant to treatment with chemotherapy. They have also been present prior to treatment with tyrosine-kinase inhibitors (ITK). The tyrosine kinase inhibitor therapy selects these cells, which enriched with CSC markers, leading to acquiring resistance. They show a high level of the OCT3/4, NANOG, SOX2 expression. They are chemo-resistant, which is characterized by determining the viability and spheres formation.      

Normally only normal stem cells and their progenitors express EMT, which allows the cells to grow in order to generate new tissues during the embryonic period and later to restore the tissue.       

However the CSCs take over the ability of normal stem cells and express EMT, which leads to cancer persistence and dissemination in other areas.        

p53, p63, p73 regulate miARN and inhibits tumorigenesis, epithelial mesenchymal transition, CSC proliferation and metastasis (97) (Stephanie Courtois M.Hayat).       

Future therapies must target only cancer stem cells, but not also normal stem cells, an important future task, but difficult to accomplish, to find a drug to target only cancer stem cells without being toxic to normal stem cells (!!)

GLOSSARY

CHAPTER I

-mRNA MCM-1(minichromosome maintenance-1). It is the transcription factor involved in cell-type specific transcription.

-CD34. Is a surface glyco-protein, encoded by the CD34 gene and also acts as cell-cell adhesion factor. It mediates the stem cells attachment to the extracellular matrix of bone marrow or directly on stromal cells.

-CD33. Classic myeloid antigen. Mediate cell-cell adhesion. Longer acts as a receptor that inhibits the proliferation of normal stem cells and myeloid leukemia cells.

-ABCG2(ATP-binding cassette subfamily G member 2). Is also termed CDw338. It is a membrane-associated protein which has a role to carry different molecules through extra or intracellular membranes.

-OCT-4(octamer –binding transcription factor 4). Is a protein encoded by the POU5F1 gene. It is involved in the embryonic stem cell self-restoration. It is a marker of undifferentiated cells. Her expression is very strongly regulated, since an expression too high or too low causes the cell differentiation.

-SOX2 (sex determing region Y)box 2. It is a transmission factor which is essential for self-restoration or pluripotency of the undifferentiated embryonic and neural stem cells. They would induce pluripotency. Promising role in reparative medicine.

-KLF 4(kruppel-like factor 4).It is an indicator of the stem cell capacity and even of mezenchimal stem cells.

-NANOG. It regulates the molecules involved with stem properties and in the signaling pathways of the cell cycle for maintaining the pluripotency of cancer stem cells (CSC). It is a key transcription factor in maintaining the embryonic stem cell pluripotency. The Nanog subregulation leads to pluripotency markers lowering, while Nanog overexpression leads to their overregulation.

-SHH(SONIC HEDGEHOG). It is one of the three proteins of the Hedgehog family of signaling pathway that in adults controls cell division and is involved in developping some cancers.

-NOTCH. It is a signaling system very well preserved and present in most multicellular organisms. Promotes cell proliferation during neuro-genesis. It is important in cell-cell connection, which involves the regulation mechanisms that control cellular differentiation processes during embryonic and adult. The Notch pathway is deregulated in some cancers.

-PTEN(phosphatase and tensin homolog deleted on chromosome ten). It is a tumor suppressor gene that seems to negatively control the way phosphatydil-innositol 3 kinase for regulating the cell proliferation and cell survival by dephosphorylition of the phosphatydil inositol 3,4,5, triphosphate.

-WNT. The WNT proteins play an important role in embryonic development, cell differentiation and cell polarity.

-EpCAM(epithelial cell adhesion molecule) is a proteine encoded by the gene of EpCAM(CD326). It is an antigen. It is expressed in undifferentiated pluripotent stem cells. It is used as a target for immune therapy of human carcinomas.

CHAPTER II

-CD133.(Prominin). It is a transmembrane glyco-protein that is specifically localized in cellular protrusions. It is expressed in hematopoietic stem cells, endothelial progenitor cells and many other cell types.

It is considered that CD133 positive cells are a population of cancer cells in small numbers, which self renew, differentiate and can propagate the tumors when injected into immuno-depressed mice. But it is difficult to isolate a population of CD133 positive cancer stem cells.

-CD44. is a cell surface adhesion molecule involved in cell-cell interactions, cellular transport, in the growth signal transmission. Many cancer cells as well as their metastases express high levels of CD44. CD44 confers an advantage to cancer cells and can be used as target in cancer therapy. The identification of CD44 cell variants (and not in normal cells) would lead to anti-CD44 reactions restricting the neoplastic growth.

-CASPASE 3. The Caspase 3 protein is a member of the CASPASE family (cysteine-aspartic acid protease). It is a cysteine-peptidase. It has a central role in the mechanism of apoptosis.

-Sca-1. It is a member of the lymphocyte antigen 6 family. It is expressed in many potent hematopoietic stem cells, also involved in the regulation of B and T cell activation.

-CD 90(Thy-1). It is a glycophosphatidyl inositol protein. Is a cell surface protein, found as thymocit antigen. Thy-1 is a marker in a variety of stem cells. Thy-1 mediates leukocyte and monocytes adhesion to endothelial cells and fibroblasts. It has a role in pulmonary fibrosis. Striking Thy-1 in mice increases the fibrosis in the lung. It is an important marker of hematopoietic stem cell pluripotency. It is also a marker for thre mezenchymal stem cells.

-Beta-catenin is a proteine encoded by the CTNNB 1 gene. It is a subunit of the Cadherin protein. Activates as an intracellular signal transductor. It is involved in the WNT signaling pathway. The gene encoding the beta-catenin functions as an oncogene. There was an increase of beta-catenin in the basal carcinoma cells.

-VEGFR-1(vascular endotelial growth factor-1) is an important signaling protein.

Involved in vasculogenesis and angiogenesis. Stimulates mitogenesis in the endothelial cell and migration cells. The micro-vascular permeability increases.

-VLA-4. Integrin alfa4 beta1(very late antigen-1). This is a dimer. VCAM-1 (vascular cell adhesion molecule) which is located in endothelial cells, binds to the integrin VLA-4, which is normally expressed in white blood cell membranes. VLA-4 integrin is a cell surface receptor, which is involved in the interaction between the lymphoid cells and extra cellular matrix (ECM) and endothelial cells.

-cMYC. It is a regulator gene, that encodes for a transcription factor. The mutated version of cMYC mutation is observed in many cancers. It is located on chromosome 8 and regulates the expression of 15% of cell genes. In addition to being a classic transcription factor it also regulates the chromatin structure by regulating histone acetylation.

-SNAIL. Is a transcription factor expressed in fibroblasts and in some epithelial tumor cell lines, with the lack of of E-cadherin expression. Its inhibition leads to restoring the E-CADHERINE expression.

-TGF-BETA. This is a protein that controls cell proliferation, cell differentiation. This is a type of cytochină, which plays a role in cancer, immunity. TGF-beta acts as an anti-proliferative factor in normal epithelial cells and in early stages of oncogenesis. It stops the cell cycle at G1 phase and stops proliferation, differentiation and apoptosis. However, due to mutations, increases thr cancer invasiveness.

- STEM CELL CLASSIFICATION.

Stem cells are classified according to their plasticity and capacity to develop:        

- allpotent stem cells. Have the potential to generate all cells and tissues.       

- pluripotent stem cells. They can give rise to several types of cells and tissues, but can not give rise to a whole organism.        

- unipotent stem-cells. Are cells that give rise to a single type of cell in tissue.

CHAPTER III CANCER STEM CELLS AND LUNG CANCER

-BASC(bronchioalveolar stem cells). They are located at the junction of the bronchio-alveolar duct. They are resistant to aggression, self-recovery and proliferation capacity. They are multipotent stem cells. They maintain the CLARA bronchio-alveolar cells and epithelial alveolar cells in the distal lung. Have been identified and characterized as CD34 positive -Sca-1positive and CD45-PECAM negative express the CCSP (Clara cell secretion protein) and the pro-surfactant protein C, which are markers of Clara cells, and epithelial - alveolar type II cells. they develop under the action of Kras oncogene.

-PECAM-1 (platelet endothelial cell adhesion molecule-1) or CD34. It is encoded by the PECAM gene localizată in chromosome17. This is an adhesion molecule of platelets and endothelial cells. It is part of a superfamily of immuno-globulins. It is involved in cell migration, the angiogenesis, activation of integrins.

-CCSP. CLARA cells protect the brochiolar epithelium. It performs this by secreting products, including CCSP and a solution similar to surfactant component. The CCSP has also an anti-inflammatory role and intervenes on the immuno-shaping functions in the lung. CCSP is secreted by non-ciliated epithelial cells.

-CD24(signal transducer 24). It is a proteine encoded by the CD 24 gene. Is an adhesion molecule.It is expressed in most B lymphocytes.

-CD29 is a cell adhesion molecule (AKA integrin beta-1).

-CD45 is a common leukocyte antigen. It is a type I transmembrane protein, present in hematopoietic cells except erythrocytes.

-CD87 It is a urokinase activator receptor-type plasminogen. It is present in small cell lung cancer.

-CD326(EpCAM). It is an adhesion molecule. It is expressed in epithelial cells and some tumors. Is used as anti-tumor target. It is expressed in pluripotent undifferentiated cells.

-Cyto-keratin. It is a protein which contains keratin, and is found in the intra-cytoplasmic cyto-skeleton of the epithelial tissue.

-VIMENTIN. Is a protein that is expressed in the mezenchymal cells. It is a marker of the mesenchyme-derived cells and cells undergoing the epithelial-mesenchymal transition both in the normal period and in tumor progression.

p53, p63, p73 are a tumor-suppressor gene family.

.

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