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A growing body of experimental evidence has revealed that the highly tumorigenic cancer stem/progenitor cells endowed with stem cell-like properties might be responsible for initiation and progression of numerous aggressive epithelial cancers into locally invasive, metastatic and incurable disease states. The malignant transformation of tissue-resident adult stem/progenitor cells or their progenies into tumorigenic and migrating cancer stem/progenitor cells and their resistance to current cancer therapies have been associated with their high expression levels of specific oncogenic products and drug resistance-associated molecules. In this regard, we describe the tumorigenic cascades that are frequently activated in cancer stem/progenitor cells versus their differentiated progenies during the early and late stages of the epithelial cancer progression. The emphasis is on the growth factor signaling pathways involved in the malignant behavior of prostate and pancreatic cancer stem/progenitor cells and their progenies. Of clinical interest, the potential molecular therapeutic targets to eradicate the tumor- and metastasis-initiating cells and their progenies and develop new effective combination therapies against locally advanced and metastatic epithelial cancers are also described.
Recent progresses in cancer research have indicated that the most human tumors including the epithelial cancers, might arise from the malignant transformation of tissue-resident adult stem cells and/or their progenitors into tumorigenic cancer stem/progenitor cells, also designated as cancer- and tumor-initiating cells (Fig. 1) [1–27]. The epithelial cancers harboring a small subpopulation of cancer stem/progenitor cells include lung, skin, head and neck, thyroid, cervical, renal, hepatic, esophageal, gastrointestinal, colorectal, bladder, pancreatic, prostate, mammary and ovarian cancers [1–27]. More specifically, it has been shown that the immature cancer stem/progenitor cells isolated from primary patient’s tumors or established cancer cell lines, which possess the capacity to self-renew and aberrant differentiation ability were able to give rise to the bulk mass of more differentiated cancer cells in vitro, and to reconstitute the patient original cancer-like tumors in mice in vivo [1–27].
In addition, the accumulation of genetic and/or epigenetic alterations resulting in the sustained activation of diverse developmental cascades in cancer stem/progenitors may also lead to their acquisition of a more malignant behavior along cancer progression (Fig. 1) [11,12,14,25,28–42]. The tumorigenic cascades include hedgehog, epidermal growth factor (EGFR), Wnt/β-catenin, Notch, stem cell factor (SCF)/KIT, bone morphogenic proteins (BMPs) and/or stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) (Fig. 2) [14,17,21,25,30,31,36,37,41–51]. Especially, the acquisition of a migratory phenotype by tumorigenic cancer stem/progenitor cells during epithelial-mesenchymal transition (EMT) may promote the development of more invasive and metastatic epithelial cancers [12,25,28,35,36,45,52–62]. In fact, the tumorigenic and migrating cancer stem/progenitor cells, also termed as metastasis-initiating cells, can evade the primary malignant neoplasm, migrate via the peripheral circulation at distant tissues and organs, and thereby contribute to metastasis formation by giving rise to a heterogeneous cancer cell population at secondary sites (Fig. 1). In support with this concept of carcinogenesis, the immature tumorigenic cancer cells with stem cell-like properties have been identified in peripheral blood and metastatic tissues of cancer patients and cell lines [3,4,11,12,15,18,35,50,52,63–72].
Importantly, it has also been shown that the cancer stem/progenitor cells may be more resistant than their differentiated progenies to current anti-cancer therapies, and thereby contribute to the tumor re-growth and disease relapse [2, 12, 14, 15, 17, 24, 27, 29,33,37,60,68,73–84]. Thus, on the basis of these observations, it appears that the elimination of this minority of tumor- and metastasis-initiating cells, which are responsible of the primary tumor formation and metastases at distant tissues, is essential for the development of more effective curative treatments against aggressive and recurrent epithelial cancers. Therefore, the use of the combinations of drugs that are able to induce the cytotoxic effects on cancer stem/progenitor cells and their more differentiated progenies should eliminate the total cancer cell mass, and thereby lead to a more complete remission without chance of relapse.
In this context, we describe the recent advancements on the establishment of deregulated signaling cascades, which are frequently associated with the malignant transformation of tissue-resident adult stem/progenitor cells into tumorigenic, migrating and metastatic cancer progenitor cell subpopulations during epithelial cancer progression. Of clinical interest, we also describe new therapeutic strategies for the development of more effective treatments against aggressive epithelial cancers by molecular targeting cancer stem/progenitor cells and their differentiated progenies as well as their local microenvironment.
The cancer initiation is generally associated with the inactivation of distinct tumor suppressor gene products such p53, p16INK4A and retinoblastoma (Rb) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and occurrence of oncogenic events in tissue-resident adult stem/progenitor cells and/or their more committed progenies (Fig. 1) [7,17,31,48,49,85–91]. These genomic alterations include the mutations, deletions, amplifications, chromosomal rearrangements and changes in DNA methylation [7,17,31,48,49,85–91]. Particularly, cumulative telomere shortening, sustained oxidative stress, chronic inflammatory and/or fibrosis, occurring during chronological aging may culminate to a genomic instability and malignant transformation of these immature cells [7,17,31,42,48,49,85–94]. Moreover, the persistent activation of diverse developmental cascades frequently occurs in the cancer stem/progenitor cells and their progenies during the etiopathogenesis and progression of localized cancers to aggressive and metastatic disease states [14,17,21,25,30,31,36,37,41–48,50]. Therefore, the molecular targeting of deregulated signaling elements in tumor- and metastasis-initiating cells is of major interest to counteract the epithelial cancer development and improve the current cancer therapies.
Several current therapies used in the clinics for the treatment of aggressive, localized advanced and metastatic epithelial cancers are based on the use of drugs showing antitumorigenic properties and which are able to eliminate cancer cells. However, these treatments generally target the global tumor cell population and no distinction is usually made between highly tumorigenic cancer stem/progenitor cells and their more differentiated progenies (Fig. 1). Therefore, despite the combinations of diverse strategies by using surgery, radiotherapy, anti-hormonal therapy and/or chemotherapy might be effective for eliminating the bulk mass of differentiated cancer cells, the possibility that the cancer stem/progenitor cells with stem cell-like properties can resist to these treatment types must also be considered.
Indeed, numerous accumulating lines of evidence have revealed that the unique intrinsic and extrinsic properties of the cancer stem/progenitor cells versus their more committed and differentiated progenies may contribute to treatment resistance and disease relapse [2,12,14,15,17,24,27,29,33,37,60,68,73–84]. Similar to normal tissue-resident adult stem/progenitor cells, cancer stem/progenitor cells principally exist under a quiescent or slow-cycling and less metabolically active state [17,46,78,95–97]. Thereby, these immature cancer cells may be more resistant than their differentiated progenies to chemo- and/or radiotherapies targeting proliferative cancer cells. Moreover, the cancer stem/progenitor cells typically express high levels of diverse anti-apoptotic factors such as Bcl-2, ATP-binding cassette (ABC) multidrug transporters, including ABCG2 and multidrug resistance-1 (MDR-1) encoding p-glycoprotein (P-gp) and DNA repair and detoxifying enzymes such as aldehyde dehydrogenase (ALDH) [2,14,17,26,37,47,78,81–84,89,97–107]. Thus, in considering the fact that the resistance of locally invasive and metastatic cancers to current therapies represents one of the major causes of cancer related deaths, the molecular targeting of tumor- and metastasis-initiating cells is of great clinical interest [17,45–49,78,97,103,108]. In this regard, we reviewed novel targeting strategies designed to eradicate the total tumor cell mass consisting of tumorigenic and migrating cancer stem/progenitor cells and their progenies, and which could be used for improving the current treatments against aggressive and recurrent epithelial cancers.
The molecular targeting of stem cell-like markers and deregulated signaling elements that mediate the transforming events occurring in tumorigenic and migrating cancer stem/progenitor cells and their differentiated progenies during cancer progression represents new promising therapeutic strategies to improve the current cancer therapies [17,45–49,78,103,109,110]. Among the potential molecular targets often altered in tumor- and metastasis-initiating cells and their differentiated progenies during cancer progression, there are diverse developmental signaling pathways. These deregulated pathways include hedgehog, EGFR, ErbB2 “HER2”, Wnt/β-catenin, Notch, hyaluronan (HA)/CD44, interleukin-6 (IL-6)/IL-6R, BMI-1, stem cell factor (SCF)/KIT, extracellular matrix (ECM) component/integrin and/or SDF-1/CXCR4 signaling elements (Fig. 2 and Table 1) [11,17,45–49,51,97,103,106,111–126]. It has been reported that the blockade of these tumorigenic pathways by using a monoclonal antibody (mAb), antisense oligonucleotides (As) or a specific inhibitor or antagonist, led to a growth inhibition, a reduction of invasiveness or metastatic spread and/or apoptotic death of cancer cells with stem cell-like properties in vitro or in animal models in vivo (Fig. 2 and Table 1) [11,17,45–49,97,103,111–121,127–129].
Other potential molecular therapeutic targets also comprise the gene products that are frequently involved in sustained growth, enhanced survival and invasion during the EMT process and/or drug resistance of cancer stem/progenitor cells and their differentiated progenies [24,45,48,49,56,78,98,106,116,130–134]. These cellular signaling effectors include telomerase reverse transcriptase (TERT), Cripto-1, tenacin C, nuclear factor-kappaB (NF-kB), phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR, interleukin-4 (IL-4)/IL-4Rα, Bcl-2, survivin, snail, slug, twist, ABC multidrug efflux pumps and/or ALDH [24,45,48,49,56,78,97,98,106,116,130–136]. Importantly, some recent investigations have also indicated the potential benefit to target these signaling elements to prevent tumor growth and/or overcoming MDR phenotype and radioresistance of cancer stem/progenitor cells and their progenies (Fig. 2 and Table 2) [15,17,24,62,77,78,83,103,106,137–140].
More particularly, it has been shown that the inhibition of ALDH activity by using ALDH1-specific inhibitor, diethylaminobenzaldehyde (DEAB) or ALDH1A-targeted small hairpin RNA (shRNA) enhanced the sensibility of ESA+/CD44+/CD166+ colorectal cancer stem cells and their progenies to the active metabolite (4-HC) of cyclophoshamide (CPA) in vitro and in vivo . Moreover, in view of fact that the enzymatic catalytic subunit, TERT, which is involved in telomere maintenance, is re-expressed in the majority of human tumors including in tumor-initiating cells, numerous strategies have been developed based on its molecular targeting. Among them, there are the use of TERT-specific oncolytic adenoviruses, gene therapy and cancer immunotherapy (Figs. 1 and and2)2) [97,135,141]. For instance, it has been observed that the adoptive cell therapy (ACT) with human TERT-specific T lymphocytes inhibited tumor growth and/or metastatic spread of a variety of human cancer cell models in vivo and resulted in a decreased survival and complete cure in some mice . Interestingly, it has also been observed that the colon cancer stem cells from patient’s colon adenocarcinoama specimens did not grow in mice treated with human TERT865–873-specific T cells (Table 1) . In this regard, other attractive adjuvant therapies against aggressive and relapsed epithelial cancers also include cancer vaccines and transplantion of hematopoietic stem cells (HSCs) or activated B and T cells (Figs. 1 and and2)2) [17,47,97,142–144].
Of therapeutic interest, the induction of the differentiation of cancer stem/progenitor cells by using agents such as retinoic acid and its synthetic analogues, interferons (IFNs) or histone deacetylase inhibitor, also may represent a promising adjuvant cancer treatment [145–148]. For instance, it has been reported that the IFN-α treatment caused a dramatic reduction in the verapamil-sensitive side population (SP) cell fraction from diverse ovarian cancer cell lines . Interestingly, the results from a recent study have also revealed that the re-activation of p53 pathway in putative colon cancer stem cells by using ellipticine improved the cytotoxic effects induced by chemotherapeutic drug, 5-fluorouracil . In the same way, a restoration of microRNA (miRNA), miR-34 in human gastric cancer cells, which acts a downstream signaling element in the p53 pathway by modulating its potential targets Bcl-2, Notch, and HMGA2, has also been shown to chemosensitize cancer cells and inhibit tumor formation and growth . In fact, it has been suggested that miR-34 tumor suppressor can reduce the self-renewal ability and survival of gastric cancer stem cells .
In addition, numerous therapeutic approaches have also been developed for a selective delivery of cytotoxic or anti-angiogenic agents and mAbs in cancer cells including cancer stem/progenitor cells. These strategies comprise the use of genetically-modified stem cell-based delivery and nanoparticule- and liposome-encapsuled drug delivery (Figs. 1 and and2)2) [97,151–154]. For instance, it has been shown that human neural stem cells (NSCs) endowed with intrinsic tumor-tropic properties and genetically-engineered to secrete anti-HER2 immunoglobulin molecules can be used to selectively delivery the therapeutic antibodies to breast cancer cells in vitro and in vivo, and thereby inhibit their growth . In the same way, the use of genetically-modified endothelial progenitor cells (EPCs) or mesenchymal stem cells (MSCs), which are actively recruited to tumor stroma and participate to tumor growth, vascularization and/or metastases also may constitute the potential vehicles for a selective delivery of anti-carcinogenic agents within tumors [153,155–157]. In this matter, we are reporting, in a more detailed manner, recent advancements in the establishment of deregulated signaling elements that can contribute to the malignant behavior and treatment resistance of prostate and pancreatic cancer stem/progenitor cells and their progenies and the potential therapeutic targets for improving current cancer treatments.
Numerous factors may influence the risk of developing a prostate cancer. The precise etiological causes responsible for prostate cancer initiation and progression to androgen-independent (AI) and metastatic stages, treatment resistance and disease relapse remain not well established. In regard with this, recent studies have revealed that the accumulation of genetic and/or epigenetic alterations occurring in prostate-resident adult stem/progenitor cells during the lifespan, combined with the changes in their local microenvironment, may result in their malignant transformation into tumorigenic prostate cancer stem/progenitor cells, also designated as prostate cancer-initiating cells [16,17,46,48,115,158–171]. Moreover, the acquisition of a more malignant behavior, including a migratory ability during the EMT program by tumorigenic prostate cancer stem/progenitor cells, may lead to their invasion and dissemination through circulation and metastasis at distant tissues, including bone marrow, brain, lung and liver (Fig. 1) [17, 45,46,78,158,162,172–174].
This model of prostate carcinogenesis is well supported by the identification of a rare subpopulation of human prostate cancer stem/progenitor cells comprising about 0.1–3% of the total tumor cell mass in malignant prostatic adenocarcinomas and metastatic neoplasms [16,163,164,168,175–177]. More specifically, the results from immunohistochemical analyses performed on malignant primary prostate adenocarcinoma specimens from patients have revealed that a small subpopulation of basal or intermediate androgen receptor (AR−) prostate cancer cells expressed the CD133 stem cell-like marker [48,164,168,175,178]. In contrast, it has been noticed that the luminal AR+ prostate cancer cells detected in same tissue samples did not express the CD133 protein [48,164,168,175,178]. Moreover, the data from the microarray studies carried out with clinical prostate cancer tissue specimens have also revealed that the transforming growth factor-β receptor 3 (TGFBR3) down-regulation, which is an important step in prostate tumorigenesis, was accompanied by an increase in the CD133 expression level . Similarly, the CD44 stem cell-like surface marker has also been detected in basal cells in normal prostate tissues and only in a minority of prostate cancer cells in certain cases of low- and high-grade prostate adenocarcinoma tissues [161,179,180].
Importantly, the prostate cancer stem/progenitor cells expressing different prostate stem cell-like markers such as CD133, CD44, α2β1+/high-integrin, cytokeratin (CK5/14), CK18, CXCR4 and/or multidrug transporters have also been isolated from primary and metastatic prostate cancers and established prostate cancer cell lines [107,163,164,168,175–177,182]. These isolated AR−/low prostate cancer stem/progenitor cells were able to give rise in vitro and in vivo to the bulk mass of differentiated cancer cells expressing luminal secretory androgen-responsive cell phenotypes, including high expression levels of AR [107,163,164,168,175–177,182]. These immature prostate cancer cells were also able to reconstitute the tumors in vivo with a histological architecture of a Gleason grade comparable to the patient’s original tumors. For instance, CD133/α2β1-integrin+/high prostate cancer stem cells isolated from primary prostate neoplasm (P4E6), when grafted orthotopically in a matrigel plug containing human prostate stroma, formed multiple intraprostatic tumors in nude mice in vivo, showing a histology like the original Gleason 4 grade prostate cancer . Moreover, it has been shown that certain established human prostate cancer cell lines may represent a heterogeneous population of prostate cancer cells, and the presence of a small subpopulation of tumorigenic prostate cancer stem/progenitor cells expressing stem cell-like markers may be responsible for their capacity to form tumors and metastasize in animal models in vivo with a high occurrence [64–66,169,183].
Thus, on the basis of these observations, it appears likely that only a small number of tumorigenic prostate cancer stem/progenitor cells and/or their early progenies with stem cell-like properties might be necessary to drive tumor growth in the prostate gland and metastases at distant tissues by giving rise to the total tumor cell mass [16,17,45,46,48,162–170,175,176,184–186]. Moreover, the occurrence of distinct genetic and/or epigenetic alterations leading to a deregulated expression and/or activity of different gene products in prostate cancer stem/progenitor cells and their differentiated progenies, and more particularly during EMT process, may culminate in more aggressive prostate cancer subtypes, treatment resistance and disease relapse.
Particularly, the aberrant expression and/or activity of diverse hormones, growth factors, cytokines and/or their receptors may lead to the stimulation of tumorigenic signaling elements that contribute to the sustained growth, survival and invasion of tumor cells during prostate cancer etiology and progression (Figs. 1 and and2)2) [48,118, 158,161,166,187–198]. Among them, EGFR, sonic hedgehog, Wnt/β-catenin, HA/CD44 and/or SDF-1/CXCR4 cascades appear to provide critical roles for the malignant transformation of prostate cancer stem/progenitor cells and their progenies, during disease progression as well as for the resistance to current clinical therapies and disease relapse [48,118,158,161,187,189,193–197]. Moreover, the high expression and/or activity of telomerase, ABC multidrug transporters including ABCG2 and MDR-1 encoding P-gp, NF-kB and PI3K/Akt/mTOR signaling elements also may contribute to the malignant behavior, survival and chemoresistance of prostate cancer- and metastasis-initiating cells and their progenies [48, 106,115,182,199].
Prostate cancer is the most common malignancy and the second leading cause of cancer-related deaths in men in the United States [48,158,166,190–192,200–206]. Although progress in developing early detection tests has led to improved clinical treatments of patients diagnosed with low-grade and organ-confined prostate cancers by radical prostatectomy, radiotherapy and/or anti-hormonal therapy, the progression to locally advanced, invasive and metastatic hormone-refractory prostate cancers (HRPCs) usually leads to disease relapse [48,158,166,190–192,200–203,205–207]. In fact, despite the patients diagnosed with localized prostate cancer initially respond to androgen deprivation, AI lesions may eventually develop and progress despite low levels of circulating androgens. This hormone-refractory disease is resistant to conventional treatments by anti-hormonal therapy, radiotherapy and chemotherapy [48,158,166,190–192,201,202,204,205,208].
More specifically, the first-line systemic docetaxel-based chemotherapies used as care for patients with high-risk or metastatic HRPCs are only palliative and typically culminate in the death of patients after about 12 to 19 months [48,158,191,192,201–203,205,208,209]. Moreover, at present time, there are no proven targeting approaches and effective therapeutic regimens for treating high-risk localized, metastatic and relapsed HRPCs. Therefore, a better understanding of the etiological causes responsible for prostate cancer etiopathogenesis and progression to aggressive and AI disease states and resistance to current androgen ablation and chemotherapeutic treatments is highly needed.
Importantly, a growing body of experimental evidence has revealed that highly tumorigenic and/or migrating prostate cancer stem/progenitor cells endowed with high self-renewal ability and aberrant differentiation potential can provide critical functions in disease progression, metastases at distant tissues, resistance to current therapies and cancer relapse [16,17,162,165–170,176,184–186,210,211]. Especially, the intrinsic properties of prostate cancer stem/progenitor cells as well as their acquisition of a more malignant phenotype during disease progression may give to them the survival advantages, and thereby contribute to their resistance to current therapies [45,46,158,212]. Consequently, most patients who undergo potential curative treatments for locally advanced prostate cancers and/or disseminated disease stages may subsequently relapse due to the persistence of AR− prostate cancer stem/progenitor cells and/or their early AI progenies in primary neoplasms and/or micrometastases at distant sites [14,17,26,30–32,36,39,40,45,46,98, 99,130–132,158,163,212–218]. Thus, it appears that the molecular targeting of highly tumorigenic prostate cancer- and metastasis-initiating cells may represent a promising strategy for improving current therapies and prevent disease relapse.
Recent studies have led to the identification of diverse signaling elements in the prostate cancer stem/progenitor cells and their progenies that could be targeted to eradicate the total tumor cell mass. Thereby, these novel potential molecular therapeutic targets could be exploited to reverse treatment resistance and improve the efficacy of current anti-androgenic treatments and docetaxel-based therapies against locally advanced, AI and metastatic prostate cancers.
As a matter of fact, it has been observed that the treatment of human parental CWR22RV1 and PC3 prostate cancer cell line-derived xenografts with an inhibitor of smoothened (SMO) co-receptor of hedgehog cascade, cyclopamine resulted in a complete regression of the in vivo tumor growth without sign of disease recurrence after 58 and 148 days of treatment cessation, respectively . These data suggest that the prostate cancer-initiating cells may be eliminated by this treatment type. It has also been noticed that the normal prostate epithelial cells were insensitive to the cytotoxic effects of cyclopamine .
Of clinical interest, we have also observed that a combination of cyclopamine, EGFR tyrosine kinase activity inhibitor, gefitinib and chemotherapeutic drug, mitoxantrone, alone or in combination, induced an arrest of growth and cytotoxic effects on parental metastatic DU145 and PC3 cells and CD44+/high and CD44−/low fractions isolated from these prostate cancer cell lines . Importantly, the results from our recent works have also revealed that an increase of the expression levels of EGFR and sonic hedgehog signaling elements occurred in a small subpopulation of CD133+ prostate cancer stem/progenitor cells and the bulk mass of CD133− cells in certain cases of patients relative to normal prostate tissues . We have shown that these tumorigenic cascades may contribute to the malignant behavior of CD133+ and CD133− cancer cell fractions from invasive WPE1-NB26 and metastatic PC3 cell lines . Of therapeutic interest, we have also observed that the CD133+ SP cells endowed with stem cell-like properties isolated from the WPE1-NB26 cell line were insensitive to a treatment with 2 nM docetaxel . This docetaxel concentration, however, induced significant anti-proliferative and apoptotic effects on the CD133− non-SP cell fraction . In contrast, cyclopamine and gefitinib were effective to induce the anti-proliferative and cytotoxic effects on both the isolated CD133+ SP cells and CD133− non-SP cell fractions . Interestingly, a combination of low concentrations of docetaxel plus cyclopamine and gefitinib also induced greater anti-proliferative and apoptotic effects on CD133+ SP and CD133− non-SP cell fractions than individual drugs or two drug combinations . Hence, these data suggest that the targeting hedgehog and/or EGFR may constitute a potential strategy for reversing the resistance of prostate cancer cells with stem cell-like properties to current chemotherapeutic drug, docetaxel.
In addition, it has also been reported that the parthenolide (PTL) a sesquiterpene lactone from the plant feverfew, induced the cytotoxic effects on parental and CD44high and CD44−/low cancer cell fractions isolated from prostate cancer cell lines, DU145, PC3, VCAP and LAPC4, as well as primary prostate cancer cells from patient samples in vitro through an inhibition of src-related signaling components . PTL was also effective to inhibit the tumor growth of CD44high DU145 cell xenograft models in vivo . Furthermore, PTL, which can act on different pathways including NF-kB, also induced the cytotoxic effects on the CD133+ primary prostate tumor cells while the CD133+ normal cells from benign prostate hyperplasia, BPH were insensitive to this treatment type in vitro .
On the other hand, it has been shown that the activation of PI3K/Akt/Fox03a signaling cascade may contribute to the prostasphere formation and maintenance of PTEN-positive DU145 and PTEN-negative PC3 cells . The treatment of these prostate cancer cell lines with PI3K inhibitor, LY294002 or dual PI3K/mTOR inhibitor NVP-BEZ235 was accompanied by a growth inhibition and cytotoxic effects on bulk mass and CD133+/CD44+ cell fraction detected in these prostate cancer cells by cytometric analyses . Additional investigations are however necessary to validate these potential therapeutic targets on distinct prostate cancer stem/progenitor cell models in vivo and in clinical trials.
Although the precise causes responsible for pancreatic cancer development are not well known, a growing body of experimental evidence suggests that the pancreas carcinogenesis could derive from the malignant transformation of pancreatic stem/progenitor cells and/or their early progenies with stem cell-like properties into pancreatic cancer stem/progenitor cells [12,49,116,221–224]. In fact, the formation of pancreatic intraepithelial neoplasias (PanINs) has been associated with the occurrence of specific genetic mutations and/or sustained activation of growth factor cascades in a small subpopulation of pancreatic stem/progenitor cells with a ductal and/or acinar cell-like phenotype localized within the exocrine compartment of adult pancreas [49,116,221,225].
In addition, this model of pancreas carcinogenesis also implicates that the PanIN formation and cancer progression into locally invasive and metastatic disease stages, may represent a continuum resulting from the accumulation of different genetic and/or epigenetic alterations in pancreatic cancer stem/progenitor cells and their progenies. These oncogenic events may lead to their acquisition of more malignant phenotypes, including a migratory ability (Fig. 1) [12,49]. Consistent with the major implication of highly tumorigenic and migrating pancreatic cancer stem/progenitor cells in tumor growth and metastases at distant tissues, a small subpopulation of immature cells with stem cell-like properties has been identified and isolated from primary and metastatic pancreatic cancer tissue specimens and established cell lines [12,17,223,226–228]. The isolated pancreatic cancer stem/progenitor cells, also designated as pancreatic cancer- and metastasis-initiating cells, expressed several stem cell-like markers, such as CD133, CD44, CD24, ALDH, ABCG2 transporter and/or CXCR4 [12,17,49,223,226–228]. The highly tumorigenic pancreatic cancer stem/progenitor cells endowed with a high self-renewal potential and aberrant differentiation potential were able to give rise to the bulk mass of differentiated pancreatic cancer cells in vitro and to reconstitute the patient’s original tumors in animal model in vivo [12,49,223,227]. Of particular clinical interest, recent accumulating lines of evidence have also revealed that highly tumorigenic and migrating pancreatic cancer stem/progenitor cells may be more resistant to current chemotherapies, and thereby provide critical functions in tumor re-growth, metastases at distant tissues after treatment initiation and disease relapse [12,129,228].
Pancreatic cancer is a highly aggressive and lethal malignancy with a poor prognosis and a long-term overall five-year survival rate less than 5% for patients diagnosed with locally advanced and metastatic disease stages [49,204,224,229–233]. Although the gemcitabine-based chemotherapeutic treatments represent the standard of care for treating the patients with locally advanced and metastatic pancreatic cancers in the clinics, these regimens are only palliative and generally result in drug resistance, disease relapse and the death of cancer patients [49,224,229–232]. This inefficacy of current chemotherapies for treating patients diagnosed with pancreatic cancers underlines the critical importance to delineate the molecular mechanisms that may contribute to chemoresistance of pancreatic cancer cells and validate new molecular therapeutic targets.
Importantly, the intrinsic properties of pancreatic cancer stem/progenitor cells as well as their acquisition of more malignant phenotypes during disease progression may notably provide them with survival advantages, and thereby contribute to their resistance to current therapies and disease relapse [12,49,129,228,233–235]. In support with this assumption, it has been observed that the treatment of pancreatic cancer cells with current chemotherapeutic drug, gemcitabine led to an enrichment in the number of cancer cells with the stem cell-like properties in vitro and in vivo [12,129,228,233–235]. More specifically, it has been shown that a small subpopulation of CD133+ pancreatic cancer cells with stem cell-like properties isolated from patient’s malignant primary neoplasm was more resistance to gemcitabine than the CD133− pancreatic cancer cell fraction . Moreover, it has also been observed that the CD133+ L3.6pl cells were more resistance to gemcitabine treatment as compared to the CD133− L3.6pl cells in vitro and an enrichment of CD133+ L3.6pl cell fraction in total tumor cell mass also occurred after gemcitabine treatment relative to the control in mice in vivo . In the same way, the gemcitabine treatment of pancreatic tumor xenografts in vivo also resulted in an enrichment in a subpopulation of pancreatic cells expressing a high level of stem cell-like markers such as ALDH and CD24 .
Thus, based on together these observations, it is likely that the patients with locally advanced pancreatic cancers and/or disseminated disease stages who undergo gemcitabine-based chemotherapies may subsequently relapse due to the persistence of chemoresistant pancreatic cancer stem/progenitor cells in primary neoplasms and/or micrometastases at distant tissues. Hence, these studies underlines the clinical interest to also considering the molecular targeting of pancreatic cancer stem/progenitor cells for overcoming treatment resistance, and thereby prevent tumor re-growth and disease relapse.
Recent investigations have led to the identification of potential molecular targets for eradicating the pancreatic cancer- and metastasis-initiating cells and improving the efficacy of current chemotherapeutic drug, gemcitabine. For instance, it has been observed that the treatment of human E3LZ10.7 pancreatic cancer cell line with SMO inhibitor, cyclopamine resulted in a reduction in the number of putative ALDH-expressing progenitor cells in vitro as well as an inhibition of metastases of E3LZ10.7 cells in an orthotopic xenograft model in vivo . In the same way, the treatment of E3LZ10.7 pancreatic cancer cell line with another SMO inhibitor type, orally bioavailable small-molecule IPI-269609, also reduced the cancer cell fraction with high ALDH activity detected in primary tumors established from orthotopic implantation in vivo . The IPI-269609 treatment also inhibited the systemic metastases of E3LZ10.7 cells in an orthotopic xenograft model in vivo . Moreover, it has been reported that a combination of cyclopamine and gemcitabine induced a tumor regression and decrease in cancer stem cell-like marker expression such as CD24 and ALDH detected in an in vivo xenograft model established from human Panc185 pancreatic cells .
Importantly, the restoration of miR-34 expression in p53-mutant human pancreatic cancer cell lines, MiaPaCa-2 and BxPC3, by either transfection of miR-34 mimics or infection with lentiviral miR-34-MIF has also been observed to result in a down-regulation of its targets, Bcl-2 and Notch1/2 . The enhanced miR-34 expression was accompanied by an inhibition of the clonogenic cell growth and invasion, induction of apoptosis and enhanced sensitivity of pancreatic cancer cells to radiation and chemotherapeutic treatment . Of therapeutic interest, the miR-34 restoration in CD44+/CD133+ MiaPaCa2 cell fraction expressing high levels of Notch/Bcl-2, and loss of miR-34 also led to an 87% reduction in the number of tumor-initiating cell population and a significant inhibition of their tumorsphere growth in vitro and tumor formation in vivo .
Numerous studies have also indicated that the enhanced expression and activation of different oncogenic products in pancreatic cancer-initiating cells and their differentiated progenies during EMT process may confer to them more malignant phenotypes and survival advantages, including a migratory ability and gemcitabine-resistant phenotype along cancer progression. Therefore, the molecular targeting of these altered gene products induced in pancreatic cancer cells, including tumor-initiating cells during EMT process is of immense clinical interest to prevent the metastases and disease relapse (Fig. 1). In this regard, our recent studies have indicated that the MUC4 mucin is expressed in CD133+ pancreatic cancer cells and the CD133− cells constituting the bulk tumor mass during disease progression . Of therapeutic interest, we have observed that the MUC4 down-regulation reversed gemcitabine resistance of parental metastatic CD18/HPAC pancreatic cancer cells as well as the CD133+/CD44high SP and CD133−/CD44low non-SP cell fractions detected in total CD18/HPAC cancer cells . In this context, it has also been reported that the treatment of HPAC and CFPAC-1 pancreatic cancer cells with high-dose of gemcitabine resulted in an enrichment of gemcitabine-resistant CD44+ cells showing greater tumorigenic ability in vitro and in vivo, and sphere-forming activity than parental cancer cell lines . The expression levels of ABC transporters, including ABCB1 (MDR-1) was also enhanced in the pancreatic cell lines made resistant to gemcitabine . Interestingly, it has been shown that the ABC transporter inhibitor, verapamil re-sensitized these pancreatic cancer cells to gemcitabine treatment, and CD44 interference RNA inhibited their clonogenic activity .
Other therapeutic strategies to counteract the invasion and metastatic spread of pancreatic stem/progenitor cells also include the inhibition of EMT-associated signaling elements. In this regard, it has been reported that the treatment of the highly tumorigenic SP subpopulation and the non-SP cell fraction from PANC-1 pancreatic cancer cell line with a specific inhibitor of PI3K, LY294002 or mTOR, rapamycin was accompanied by a decrease of their survival in vitro . Moreover, the treatment of the SP cell fraction from pancreatic cancer cell lines with TGF-β has also been observed to induce the expression of EMT-associated gene such as snail and matrix metalloproteinase-2 (MMP-2) while E-cadherin expression was reduced . The SP cell subpopulation also exhibited a higher invasive ability in response to TGF-β treatment, while non-SP cells did not respond to TGF-β-mediated invasion . These data suggest that the treatment of pancreatic cancer cells with anti-TGF-β mAb could constitute a potential therapeutic strategy to counteract the EMT process and invasion of pancreatic cancer cells with stem cell-like properties. In this way, it has also been reported that the Notch signaling pathway may contribute to the EMT process and its down-regulation by siRNA approach led to a partial reversal of the EMT phenotype including a decreased expression of vimentin, ZEB1, snail, slug, sail, and NF-κB . The Notch inhibition was also associated with a decrease of the invasive behavior of gemcitabine-resistant pancreatic cancer cells . Of particular interest, it has also been reported that the inhibition of SDF/CXCR4 axis by using anti-CXCR4 mAb reduced the metastatic capacity of highly metastatic human pancreatic cancer cell line L3.6 pl harboring a CD133+/CXCR4+ cancer cell subpopulation orthotopically implanted in the pancreas of nude mice in vivo .
Recent advances in basic and clinical oncology have revealed that the tumorigenic and migrating cancer stem/progenitor cells can provide critical functions in tumor formation, metastases at distant sites, treatment resistance and disease relapse. Several deregulated gene products have been identified in cancer stem/progenitor cells and their progenies as well as in their local tumor microenvironment during epithelial cancer progression. These altered gene products include different tumor suppressor gene products, oncogenes and drug resistance-associated molecules that are involved in the regulation of the self-renewal, differentiation, survival and/or treatment resistance of tumor- and metastasis-initiating cells. Importantly, it has also been shown that the molecular targeting of these deregulated gene products may constitute the potential strategies to design novel cancer treatments for reversing the MDR phenotype, improving the current cancer therapies and preventing disease recurrence. These new promising therapeutic targets may now be exploited to develop new clinical combination therapies against aggressive, metastatic, recurrent and lethal human cancers, including the most epithelial cancers.
We apologize to the researchers that have contributed to the advancements in the cancer stem/progenitor cell research and therapies and whose works have not been cited due to space limitations. The authors on this work are supported by grants from the US Department of Defense (PC04502, PC074289) and the National Institutes of Health (CA78590, CA111294, CA133774 and CA131944).