Targeted therapy aimed at cancer stem cells: Wilms’ tumor as an example
Wilms’ tumor (WT), a common renal pediatric solid tumor, serves as a model for a malignancy formed by renal precursor cells that have failed to differentiate properly. Here we review recent evidence showing that the tumors’ heterogeneous cell population contains a small fraction of cancer stem cells (CSC) identified by two markers: Neural Cell Adhesion Molecule 1 (NCAM1) expression and Aldehyde dehydrogenase 1 (ALDH1) enzymatic activity. In vivo studies show these CSCs to both self-renew and differentiate to give rise to all tumor components. Similar to other malignancies, the identification of a specific CSC fraction has allowed the examination of a novel targeted therapy, aimed at eradicating the CSC population. The loss of CSCs abolishes the tumor’s ability to sustain and propagate, hence, causing tumor degradation with minimal damage to normal tissue.
KeywordsWilms’ tumor Cancer stem cells Kidney stem cells Renal progenitor cells Targeted therapy
Cancer stem cells—past and current
The history of the CSC theory can be traced back more than 70 years. In 1941, teratocarcinomas were found to contain both differentiated and undifferentiated cells, leading to the notion that the undifferentiated cells represent multi-potent cancer cells . In 1963, over four decades ago, Bruce and Van Der Gaag were the first to suggest the existence of cancer initiating cells (CICs) in murine lymphoma and a method for their in vivo quantification . In 1977, Hamburger et al. published a method for supporting colony growth of human tumor stem cells in soft agar [18, 19]. Buick et al. developed an in vitro system for measuring the frequency of clonogenic cells within tumors more accurately in semi-solid cultures [20, 21]. They managed to demonstrate the self-renewing ability of blast progenitors in acute myeloid leukemia (AML) [22, 23]. Consequently, McCulloch and colleagues postulated that AML can be considered as a clonal hemopathy [22, 23]. However, the first prospective identification, characterization, and isolation of CSC/CICs was performed years later in AML on the basis of their phenotypical similarities to normal hematopoietic stem cells ; in their innovative work, Dick and colleagues have identified CD34+CD38− cells as AML CSCs [24, 25]. Subsequently, the group reported that only CD34+CD38− cells were able to reproduce AML in recipient immunodeficient mice, which closely resembled the original patient’s disease, and exhibited its full heterogeneous phenotype.
Following AML, recent years have seen the identification and isolation of cancer stem cells in various solid organ malignancies. The first to identify such cells was Al-Hajj who found that breast cancer cells with CD24–CD44+ phenotype are able to form tumors that recapitulate their parental tumor when implanted in the mammary fat pad . Immediately following this discovery, CD133+ cells were identified as tumor stem cells in glioblastoma brain tumors  and thereafter in colon cancer . In the past few years, high ALDH1 activity levels have been used to identify CSCs in a variety of tumors including liver, head and neck, colorectal, breast, multiple myeloma, acute myeloid leukemia, and brain cancers [29–35]. Moreover, a link between poor prognosis and increased ALDH1 activity was found in breast tumors . Since the above discoveries, as well as additional CSCs markers, CSCs have been prospectively isolated from a variety of malignancies, thus far including pancreas, skin, head and neck, and prostate cancers, and the list is ever growing [26–28, 36–38]. The identification of CSCs was facilitated by significant progress achieved over the last several years in this field. To date, the gold standard for CSC identification is xenotransplantation of human tumor cells into immunodeficient mice. The injection of tumor cell subpopulations, selected based on the differential expression of specific markers, allows the assessment of the tumorigenic potential of different subpopulations within the tumor. The subpopulation identified with tumorigenic capacity is implicated as the CSC population. In addition, mainly for support of the in vivo methods, in vitro assays have been developed for CSC identification, including colony formation assay, sphere formation assay, the side population (SP) assay, differentiation potential assays, and label retention cell assay .
Cancer stem cells in Wilms’ tumors
Hence, the identification and characterization of the WT cancer stem cells unveiled new therapeutic targets in WT.
Wilms’ tumor treatment
Several decades ago, WT was mainly treated by means of nephrectomy and postoperative radiotherapy, with only 30 % surviving their illness . Today, most WT patients are treated with a combination of surgery and chemotherapy, while cases exhibiting poor prognostic factors are treated with radiotherapy. Reports from the National Wilms’ Tumor Studies (NWTS) identified lymph node metastases and anaplastic histology as the most significant factors predicting long-term survival . As a result of treatment protocol improvement, the 5-year overall survival for patients with WT is now over 90 % . Despite overall improved outcomes, WT treatment holds two significant challenges: tumor relapse and late adverse effects. According to the International Society of Pediatric Oncology, the relapse rate of patients is 12 %, with an overall survival of 48 % in recurrent disease . In patients without metastatic disease at presentation, approximately 75 % of all recurrences occur within 1 year after treatment completion . The prevalence of late adverse effects in long-term WT survivors is high, especially after radiotherapy and treatment with anthracyclines ; studies on survivors of childhood cancer have shown that 68 % of WT survivors had developed chronic health problems , among the most clinically significant effects are: musculoskeletal abnormalities, cardiac toxicity, reproductive problems, renal dysfunction, and the development of secondary malignant neoplasms . Great efforts are being made to improve the efficiency of WT treatments. Novel targeted treatment strategies are needed to improve clinical outcomes for children with WT as well as to reduce the toxic adverse effects of available treatment options.
Targeted therapy—targeting CSCs in WT
Anti-tumor targeted therapies are treatments aimed at specific characteristics of cancer cells that are crucial for tumor initiation and maintenance. Due to their specificity, targeted therapies are less likely to harm normal, healthy cells compared to systemic chemotherapy or radiation therapy, and therefore are expected to cause fewer side effects.
Thus far, several targeted treatments, each directed at a specific cancer trait, have been approved for clinical use. A few examples are outlined: (1) targeting of specific cell signaling pathways such as the epidermal growth factor inhibitors—cetuximab (Erbitux), a chimeric (mouse/human) monoclonal antibody (mAb), used in the treatment of colorectal cancer and head and neck carcinoma [52–55], trastuzumab (Herceptin), an anti-HER2 mAb, used against breast tumors and metastatic gastric cancer-expressing HER2 ; (2) interference with tumor angiogenesis—bevacizumab (Avastin), an anti-VEGF-A humanized mAb, used against colorectal, lung, breast, glioblastoma, kidney, and ovarian tumors [57, 58]; (3) targeting of specific tumor antigens—rituximab (MabThera), an anti-CD20 mAb, used against non Hodgkin’s lymphoma . A growing number of targeted treatments have reached the clinical setting; some replacing the conventional systemic treatments and others are used in conjunction with them to allow application of lower doses of the later, more toxic, drugs.
From a translational aspect, cancer stem cell theory predicts that CSCs should be the preferred targets of anti-cancer treatment, as they are the driving force behind tumor initiation, propagation, and recurrence [60, 61]. However, their inherent traits, which allow them to escape conventional chemo/radiotherapies, necessitate the development of alternative treatment options directed at these highly malignant and therapy-resistant cancer cells. Although therapeutics aimed at CSC eradication have not yet reached clinical use, there are several novel reports of targeted CSC therapy in animal models or in clinical trials [62, 63].
Due to tissue availability and a well-characterized cellular hierarchy of the normal hematopoietic system, the most studied CSCs are those of acute myeloid leukemia (AML), isolated over a decade ago . This discovery was followed by several efforts aimed at targeting the hematopoietic cancer stem cell markers, such as CD44 and CD123 in AML [64–68]. Further studies have since been performed by targeting CSCs in several solid tumors such as pancreatic, breast, prostate and colon cancers, melanoma, glioma, hepatocellular carcinoma, and others. These therapies are aimed at targeting a tumor-specific antigen (e.g., CD133, EpCAM, CD24 etc.) [69–71], inhibiting a signaling pathway predominantly activated in the CSCs (e.g., Notch, Wnt etc.) [72, 73], immunomodulation (e.g., CD326, ALDH1 inhibitor) [73, 74], sensitizing CSCs to systemic chemotherapy/radiation (e.g., IL4, hyaluronate receptor) [56, 75] or inhibiting CSC angiogenesis (e.g., VEGF-R, DLL4) [76–78]. An important contribution of CSC research to anti-cancer targeted treatment is that it unveils specific biomarkers which can be targeted in vivo by antibody therapy leading to disrupted tumor growth [60, 67, 79, 80]. Several of these antigens have been known to be expressed in different malignancies, long before their implication as CSC markers. However, their specific targeting was put forward as means to treat human malignancies only following the revelation of their role in signifying the CSC population [67, 81, 82].
Consequently, we found NCAM1, which has been known to be expressed in WT since the 1980s, to mark WT CSCs, hence the importance of its targeting.
The importance of targeting the WT CSCs is also supported by our data, showing that first-line chemotherapeutics used to treat WT patients do not have a prominent effect on either the NCAM1+ or NCAM1+ALDH1+ cells in vitro. The second-line course of therapy, used to treat WT patients whose disease recurred, reduces these cell populations in vitro, however, clearly does not eradicate all WT CSCs. Currently, chemotherapy regimens used to treat WT patients are employed at doses that lead to numerous adverse effects, perhaps the most feared being devastating secondary malignancies emerging about 20–30 years following treatment completion [83–86]. Taking into account that WT is usually diagnosed before 5 years of age, these effects possess an even greater impact, taking place in the patient’s early adulthood.
Altogether, NCAM, serving as a definite marker for WT CSCs, can be exploited as a therapeutic target in WT patients. Moreover, although NCAM is a renal developmental marker [87–90], human nephrogenesis completely ceases at 34 weeks of human gestation, excluding the potential for aberrant development caused by anti-NCAM treatment. Therefore, from a clinical standpoint, a combined regimen involving the specific eradication of the WT CICs via targeting of the NCAM molecule, might prove useful in reducing chemotherapy toxicity in all WT patients and particularly in those that do not respond to conventional treatment or those with recurrent disease.
Key summary points
• Wilms’ tumor (WT), the most common pediatric solid tumor of the kidney, is believed to arise from renal precursor cells that have failed to differentiate properly.
• Cancer stem cells (CSCs) are defined by two main properties: self-renewal and differentiation capacities. In recent years the CSC population has been identified in a growing number of solid and hematologic malignancies.
• NCAM1+ALDH1+ cells have been identified as the CSC fraction in WT. The capability of the tumor to grow and propagate is maintained solely by these cells.
• The use of an anti-NCAM1 antibody-drug conjugate (HuN901-DMI) results in loss of the WT CSCs, both in vitro and in vivo, causing tumor size reduction and loss of tumorigenic capacity.
Multiple choice questions (answers are provided following the reference list)
- 1.Which of the following statements regarding Wilms’ tumor epidemiology is correct?
Most patients have familial history
WT is the most frequent pediatric tumor
Two thirds of all WT cases cannot be linked to any genetic aberration
The tumor is more common among males
- 2. What is the definition of a cancer stem cell?
Activation of pluripotency genes (ie Oct4, Sox2, Nanog)
Formation of tumor spheres in low-adherence cultures
Self-renewal and differentiation capacities
- 3. Which of the following assays does not serve as a method for CSC identification and isolation?
Doubling time assay
Side population assay
Label retention cell assay
Colony formation assay
- 4.What is the common practice for WT patients?
Nephrectomy and postoperative radiotherapy
Targeted therapy aimed at cancer stem cells
A combination of surgery and chemotherapy
Conservative treatment based on low protein diet
- 5.Choose the incorrect sentence regarding the results of an anti-NCAM1 antibody-drug conjugate (HuN901-DMI)?
In vitro, the treatment resulted in depletion of the cell’s ‘stemness’ properties (CFU capacity, proliferation)
HuN901-DMI treatment presented a toxic effect, represented by mice weight loss
Treatment of mice bearing human WTs with high NCAM1 expression resulted in complete eradication of the tumors in the majority of cases
Treatment of low NCAM1 expressing WT xenograft resulted in reduction of tumor size followed by a plateau