CD117/c-kit defines a prostate CSC-like subpopulation driving progression and TKI resistance

Cancer stem-like cells (CSCs) are associated with cancer progression, metastasis, and recurrence, and may also represent a subset of circulating tumor cells (CTCs). In our prior study, CTCs in advanced prostate cancer patients were found to express CD117/c-kit in a liquid biopsy. Whether CD117 expression played an active or passive role in the aggressiveness and migration of these CTCs remained an open question. In this study, we show that CD117 expression in prostate cancer patients is associated with decreased overall and progression-free survival and that activation and phosphorylation of CD117 increases in prostate cancer patients with higher Gleason grades. To determine how CD117 expression and activation by its ligand stem cell factor (SCF, kit ligand, steel factor) alter prostate cancer aggressiveness, we used C4-2 and PC3-mm human prostate cancer cells, which contain a CD117+ subpopulation. We demonstrate that CD117+ cells display increased proliferation and migration. In prostaspheres, CD117 expression enhances sphere formation. In both 2D and 3D cultures, stemness marker gene expression is higher in CD117+ cells. Using xenograft limiting dilution assays and serial tumor initiation assays, we show that CD117+ cells represent a CSC population. Combined, these data indicate that CD117 expression potentially promotes tumor initiation and metastasis. Further, in cell lines, CD117 activation by SCF promotes faster proliferation and invasiveness, while blocking CD117 activation with tyrosine kinase inhibitors (TKIs) decreased progression in a context-dependent manner. We demonstrate that CD117 expression and activation drives prostate cancer aggressiveness through the CSC phenotype and TKI resistance.

Cell culture, sorting, and treatment. To examine how CD117 expression alters cell function, human prostate cancer C4-2 or PC3-mm cells were sorted into CD117+ and negative populations from a single passage just prior to experiments. C4-2 and PC3-mm cells were cultured in RPMI1640+10%FBS. Cells are confirmed negative for mycoplasma every six months by the WFBCCC Cell Engineering Shared Resource using the Lonza MycoAlert Mycoplasma Detection Kit. All experiments were performed within 10 passages using cells authenticated by the MD Anderson Cytogenetics and Cell Authentication Core (4/30/2019). C4-2 and PC3-mm were sorted into CD117+ and negative populations using the Miltenyi MACS sorting beads or the ThermoFisher MagniSort CD117 (c-kit) positive selection kit. Sorted cells were treated with 50 ng/mL recombinant stem cell factor (SCF; Miltenyi Biotec), 5 μM imatinib mesylate (Sigma), 5 μM sunitinib malate (Sigma), or 5 μM ISCK03 (Sigma).
IncuCyte live cell imaging. To image cells over time, an Essen Bioscience IncuCyte ZOOM live-cell imager in the WFBCCC Cell Engineering Shared Resource was used for proliferation, scratch migration, chemotaxis, and sphere formation assays. For proliferation assays, sorted cells were plated at 2000 cells per well and imaged over three days. The percentage of confluence was calculated. For scratch migration assays, sorted cells were plated at 50,000 cells per well. The next day, cells were scratched using the Essen Biosciences WoundMaker tool. Scratches were imaged for 48 h and percent wound closure measured. For chemotaxis assays, sorted cells were plated at 1000 cells/top of the insert and imaged for 48 h. The number of cells on the top and bottom of the insert were counted. For sphere formation assays, 1000 sorted cells were plated in ultra-low attachment plates and imaged over nine days. This method generates one sphere per well. The sphere area was calculated over time. Separate groups were treated with inhibitors or starved overnight before SCF treatment.
Immunofluorescence. For

Results
CD117 expression results in decreased survival. In a prior study, we demonstrated that the number of CD117+ cells circulating in prostate cancer patients was associated with cancer severity and biochemical recurrence 19 . Based on these findings, we asked how CD117 expression on these cells promoted prostate cancer progression. In an initial study, we showed that CD117+ C4-2 xenograft tumors were larger and more angiogenic than tumors containing the negative population 19 . To examine the outcomes of prostate cancer patients bearing CD117 alterations in their tumors, we used the cBioPortal database to mine the TCGA database. CD117 expression was found to be amplified in patients in 11 of the 19 studies examined representing 0.6-16.2% of the patients. Median overall survival time was reduced to 77 months in patients with CD117 alteration compared with 133 months (Logrank Test p = 5.23 × 10 -7 ) for unaltered patients (Fig. 1A). Additionally, progression-free survival was decreased in patients with CD117 gene alterations (Logrank Test p = 8.951 × 10 -3 ; Fig. 1B). We then used the Broad Institute Cell Line Encyclopedia to examine changes in CD117 expression in prostate cancer cell lines. The copy number of CD117 was not necessarily associated with mRNA and protein expression (Fig. 1C,E). The mRNA for CD117 (Fig. 1D) was highly expressed in all cell lines with lower protein expression (Fig. 1E). Accordingly, we profiled several prostate cancer cell lines including several bone-tropic lines not currently available in the Broad Institute Cell Line Encyclopedia for CD117 cell surface expression by flow cytometry. We www.nature.com/scientificreports/ discovered a small CD117+ population in PC3 (~ 0.61%), DU145 (~ 0.685%), and MDA-PCa-2b (~ 0.25%) prostate cancer cell lines (Fig. 1F). No surface CD117 expression was found on LNCaP, 22Rv1, or VCaP prostate cancer cells (Fig. 1F) despite their higher proteomic expression (Fig. 1E). The bone-tropic PC3-mm cell line contained a ~ 2% CD117+ subpopulation. The androgen-independent and bone-tropic C4-2 prostate cancer cell line (derived from LNCaP cells) contained significant CD117+ population, ~ 21.4% on average. As previously described, androgen dependent cell lines, such as LNCaP (Fig. 1F) and the LNCaP clone FCG (Fig. 1E), had no protein or surface expression of CD117 31 . We chose to examine the two bone-tropic prostate cancer cell lines with the highest surface CD117 expression, C4-2 and PC3-mm, in subsequent experiments studying the CD117+ subpopulation. For experiments requiring higher cell numbers, only the C4-2 line was studied.

CD117 expression stimulates prostate cancer proliferation and migration.
To further determine how CD117 expression drives prostate cancer aggressiveness, we sorted C4-2 and PC3-mm human prostate cancer cells into CD117+ and negative populations. Using live-cell imaging, we examined differences between these www.nature.com/scientificreports/ two populations. In a proliferation assay, CD117+ cells proliferated more quickly than the negative cells. C4-2 CD117+ cells reached confluency peak at hour 58 at which point CD1117+ cells were 2.9-fold more confluent than the negative population ( Fig. 2A). Similarly, PC3-mm CD117+ cells at hour 58 were 1.75-fold more confluent than the negative population ( Fig. 2B) Thus, in monolayer culture, CD117 promotes proliferation. Since CD117+ cells were found in the patient circulation, we examined cancer cell migration using scratch and chemotaxis assays. Confluent C4-2 and PC3-mm CD117+ and negative cells were scratched to generate a wound, and cell movement into the wound was measured by live-cell imaging. CD117+ cultures showed increased confluence within the wound and decreased wound width over time (Fig. 2C,D). Upon experimental termination, C4-2 CD117+ cell wound closure was 2.7-fold higher and PC3-mm CD117+ cell would closure was 1.2-fold higher. Additionally, C4-2 CD117+ and negative cells were plated on the top of a transwell, and migration through www.nature.com/scientificreports/ pores measured over time. At experimental termination, 3.2-fold more CD117+ cells had migrated when compared to the negative population (Fig. 2E). Thus, CD117 expression increases cell migration in two dimensions. Another marker of aggressive tumor cells is the ability to form spheres 17,[32][33][34][35][36] . Sorted cells were examined for sphere formation by live-cell imaging and changes in gene and protein expression of aggressiveness markers was measured in spheres. C4-2 CD117+ cells formed 1.35-fold larger spheres on day 5 compared with the negative cells (Fig. 3A). PC3-mm sorted cells demonstrated no differences in sphere growth (Fig. 3B). To examine aggressiveness, staining for the EMT marker vimentin demonstrated a 1.7-fold increase in C4-2 CD117+ spheres compared with negative ( Fig. 3C) and PC3-mm spheres had a 2.4-fold increase in vimentin (Fig. 3D). Additionally, a 2.0-fold increase in the stemness marker Oct4 was measured in C4-2 CD117+ spheres (Fig. 3C). To examine how growth in 3D spheres altered gene expression, we compared samples to 2D, monolayer grown cultures. The growth of C4-2 CD117+ cells in spheres upregulated Oct4 (6.8-fold) and MMP-13 (7.7-fold) compared with monolayer cultures (Fig. 3E). Interestingly, the ligand for CD117, SCF, expression was also increased with 3D growth of the C4-2 CD117+ cells. (Fig. 3E). No significant difference was seen between negative culture conditions (Fig. 3E). Taken together, these data demonstrate that CD117 expression may promote prostate cancer progression and this effect is increased when cells are grown in 3D spheres. Because the C4-2 prostate cancer  A and B). Sphere formation was tracked using livecell imaging and represented as mean sphere area (n = 12). (C and D) After 7 days, spheres were sectioned and stained for the EMT markers: E-cadherin, N-cadherin, and vimentin; and (C) the stemness markers: Sox2 and Oct4. Relative fluorescence is represented as mean ± SEM (n = 3-6). (E) Gene expression was compared between C4-2 sorted cells grown in 2D monolayer or 3D spheres and represented as mean fold change ± SEM (n = 3). *Represents p < 0.05, **represents p < 0.01, and ***represents p < 0.005 by Student's t test (A-C) and one-way ANOVA (D). www.nature.com/scientificreports/ cell line contained a higher CD117+ subpopulation, we profiled them in the rest of our studies, which required higher cell numbers.

CD117+ cells are cancer stem-like cells.
More invasive cancer cells that form prostaspheres have the potential to be CSCs 17 . Additionally, the expression of stem cell markers, including Oct3/4 and Sox2 are often used to examine stemness in potential CSCs 32,37,38 . To further demonstrate pluripotency, self-renewal capacity is measured by clonogenic assays and serial in vivo tumor initiation or limiting dilution experiments designed to examine whether a population could regenerate an entire tumor and thus be considered a CSC 13,14,16,39,40 . In order to differentiate tumor-initiating cells from CSCs, repeated tumor-initiating xenografts are required 40,41 . To determine whether C4-2 CD117+ cells displayed the CSC phenotype, we first examined the expression of stemness markers. Gene expression of Oct4 and Sox2 were 2.5-and 15.5-fold higher, respectively, in C4-2 CD117+ cells compared with the negative population (Fig. 4A). To validate if these cells were a true cancer stem-like population, we performed serial tumor initiation experiments. C4-2 CD117+ and negative cells were implanted subcutaneously in immunocompromised mice at limiting dilutions. Both populations formed tumors from 500 to 10,000 implanted cells (data not shown). At all cell concentrations, CD117+ cells formed larger tumors (Fig. 4B). At 100 cells both groups reliably formed tumors, while at 25 cells approximately 40% of CD117+ and 30% of negative cell implants formed tumors (Fig. 4C). At 10 cells, 50% (13 of 26) of CD117+ and 23% (6 of 26) of negative cell implantations formed tumors (Fig. 4C). Serial tumor initiation was completed by dissociating the 10 cell tumors, repeating bead sorting, and re-implantation at 10 cells. In serial tumor initiation, CD117+ cells formed a second tumor ~ 25% of the time (3 of 20 from 10 cells and 7 of 20 from 25 cells), while the negative implants were unable to initiate secondary tumors (data not shown). Thus, CD117 positive cells represent a prostate cancer stem-like subpopulation.

SCF activation of CD117 increases prostate cancer progression.
Based on these data demonstrating that CD117 expression drives prostate cancer progression, we questioned how activation of CD117 might alter these effects. CD117 is activated by binding to SCF, its sole ligand. SCF, found in a dimer, binds to CD117 inducing dimerization, phosphorylation, and downstream signaling leading to proliferation, cytoskeletal rear- www.nature.com/scientificreports/ rangement, and migration 21,42,43 . To examine how activation of the tyrosine kinase receptor CD117 may alter prostate cancer aggressiveness, we first examined phosphorylated (p-)CD117 levels in prostate cancer patients. The numbers of p-CD117+ cells per core increased with Gleason Score (Fig. 5A). Tumors from patients with grade 4 and 5 cancers contained 2.2-fold more p-CD117 cells compared with grade 1 patient tumors. Thus, activated CD117 is increased in patients with cancer severity. Next, we examined how CD117 activation by SCF alters prostate cancer cell aggressiveness. First, we treated our sorted C4-2 CD117+ and negative cells with SCF and found that SCF increased proliferation of CD117+ cells by live-cell imaging (1.4-fold at hour 40) but had no significant effect on the negative populations (Fig. 5B). We then tested how CD117+ cell activation by SCF would change invasion. Sorted cells were placed in a transwell system with SCF in the bottom chamber (Fig. 5C). C4-2 CD117+ cell invasion was 1.4-fold higher towards SCF than media alone at experimental termination. Thus, SCF functions as a chemoattractant factor for CD117+ cells inducing migration. Finally, SCF treatment effects on CSC gene expression was examined. CD117+ cells have much higher CSC gene expression compared with the negative population. SCF treatment stimulated small, but insignificant decreases in Oct4 and Sox2 expression in CD117+ cells and an increase in Sox2 expression only in negative cells (Fig. 5D). Taken together, these data demonstrate that CD117 activation induces prostate cancer progression as shown by proliferation and migration. CD117 inhibition prevents prostate cancer progression. Since CD117 activation induced prostate cancer progression and migration, we next examined the effects of treatment with the tyrosine kinase inhibitors (TKIs): sunitinib, imatinib, and ISCK03. Sunitinib targets several pathways and receptors including PDGFR and VEGFR in addition to CD117 21,[44][45][46] . Imatinib targets CD117, in addition to BCR-Abl, RET, and PDGFR 44,47 . ISCK03 is a cell-permeable CD117 specific inhibitor that blocks SCF-induced phosphorylation [48][49][50] . In proliferation studies, sunitinib had the greatest reduction of proliferation for CD117+ cells (85% at 70 h), while ISCK03 treatment resulted in a 50% reduction in proliferation and imatinib treatment did not affect proliferation (Fig. 6A). We next examined the effect of TKIs on growth in 3D. In contrast to proliferation in monolayer, imatinib had the strongest effect inhibiting sphere growth 66%, while sunitinib induced 50% inhibition, and ISCK03 had minimal effect at hour 136 (Fig. 6B). Taken together, these data demonstrate that TKIs decrease CD117 induced proliferation and sphere formation, but the effects of the individual TKIs are dependent on the culture conditions. Overall our data demonstrate that CD117 activation plays a key role in prostate cancer, progression, migration, and resistance to TKIs. www.nature.com/scientificreports/

Discussion
In this study, we determined how the CTC marker CD117 expression and activation affected prostate cancer progression. CD117 expression on human prostate cancer cells induced increased proliferation, migration, and sphere formation. CD117+ cells expressed stemness marker genes and generated tumors in serial tumor initiation studies indicating that CD117-expressing cells represent a CSC. Beyond expression, CD117 activation was associated with increased cancer severity. SCF activation of CD117 stimulated further proliferation and invasion but had no additional effect on CSC genes. Conversely, CD117 inhibition by TKIs diminished cell proliferation and sphere formation. Our data suggest that CD117 activation drives prostate cancer progression, invasion, and TKI resistance through its induction of the CSC phenotype. We demonstrate that CD117 expression induces prostate cancer progression, and its activation increases with cancer severity. CD117 expression and overactivation is found in several cancers including gastrointestinal stromal tumors (GIST), acute myeloid leukemia, and melanoma 21,24,[51][52][53] . CD117 is best studied in GIST, which carries CD117 activating mutations. In GIST, CD117 expression and activation is associated with worse prognosis and bone metastasis [54][55][56][57] . However, activating mutations have only been found in GIST despite increased expression in prostate and ovarian cancers among others 21,53,58 . CD117 expression in many cancers is associated with shorter disease survival and metastasis. CD117 is low in benign prostate tissues and is increased with cancer progression in prostate cancer patients with the highest levels of CD117 staining seen in bone metastases 21,31,[58][59][60] . However, the opposite is true in myeloid/erythroid cancers, whose patients have improved or unchanged prognosis when CD117 is expressed 61 . This may be partially due to biological differences between hematologic malignancies and solid tumors in which some genes and miRNA have opposite functions 62 . Further, in solid tumors, cancer cells are often the result of dedifferentiation whereas in hematological malignancies cancer cells are often transformed HSCs or from earlier lineage cells. Thus, the differences in CD117 function in these tumors may relate to the degree of "stemness. " Our data indicate that the CD117 subpopulation represents a prostate CSC. This finding is supported by data demonstrating that CD117 expressing cells in the normal prostate are also stem-like. CD117+ prostate stem cells were found in all murine prostate lobes and both the luminal and basal compartments. Prostates generated from a single CD117+ cell contained neuroendocrine synaptophysin-positive cells and expressed probasin and Nkx3.1 22 . CD117 is a well-known stem-cell marker for normal hematopoietic cells, and its expression declines as cells lose their plasticity during differentiation [63][64][65] . Further, in other cancer types, including lung and ovarian cancers, CD117 expressing cells exhibited CSC characteristics including self-renewal [66][67][68] . In osteosarcomas, CD117 is expressed on CSCs and confers resistance to chemotherapies 69 . In that study, CD117+ osteosarcoma cells formed spheres and successfully initiated tumors after serial transplant similar to our findings with the prostate cancer cell line. Further, in accordance with previous studies for prostate cancer, CD117 expression was higher in metastatic osteosarcoma tumors. Thus, CD117 expression appears to correlate with an ability to metastasize to bone. Our prior study demonstrating CD117 expression on CTCs, in combination with this study demonstrating that CD117+ cells are tumor-initiating cells, suggests that CD117 likely plays an important role in driving bone metastasis although future studies are needed to examine a causal link. CD117+ cell escape from the primary tumor could be caused by chemotaxis towards SCF as demonstrated by our in vitro data. SCF plays an important role in the homing to and maintenance of HSCs in the bone microenvironment 31,65,70,71 . Bone marrow niche cells secreting SCF include perivascular cells, endothelial cells, pericytes, mesenchymal stem cells, megakaryocytes, and stromal cells 72,73 . Additionally, osteoblasts produce SCF and control CD117 expressing HSC numbers near trabeculae 74 . Correspondingly, SCF deletion in endothelial cells or pericytes leads to HSC depletion in bone marrow 72,75,76 . SCF also plays an important role in cancer and metastasis. Bone marrow stromal cells and prostate cancer express membrane SCF and release the soluble form. However, bone marrow stromal cells express much higher levels of the soluble SCF 31 . These data indicated that SCF in the bone marrow might function as a chemoattractant stimulating prostate cancer bone metastasis as already demonstrated during Ewing's sarcoma metastasis to bone 77 . Interestingly, C4-2 prostate cancer cells contain a subpopulation of CD117+ cells, while the LNCaP parental subline does not. It is likely that the exposure Figure 6. Inhibition of CD117 decreased proliferation and sphere formation. Sorted C4-2 CD117+ cells were treated with the broad tyrosine kinase inhibitors imatinib and sunitinib, as well as, ISCK03, a CD117specific inhibitor. (A) Proliferation was measured by live-cell imaging and represented as mean percent confluence ± SEM (n = 6). (.) Sphere formation was tracked using live-cell imaging and represented as mean sphere area (n = 12). ***Represents p < 0.005 by one-way ANOVA. www.nature.com/scientificreports/ of the LNCaP cells to bone marrow during the generation of the C4-2 subline induced CD117 expression 78 . This was shown with the PC3 cells that once exposed to bone marrow cells started to express CD117 31 . In other cell types, SCF treatment can induce CD117 expression but can also induce internalization of the receptor 42,79,80 . These data may explain why SCF treatment stimulated increased Sox2 expression in the negative population and had little effect on the CD117+ cell population gene expression. Further studies are needed to examine how SCF treatment might promote CD117 expression in negative cells. Binding of SCF to the CD117+ cells may confer therapeutic resistance due to internalization of the receptor or via competition with TKIs, such as imatinib, for the CD117 activation site 81 . Our data demonstrate that CD117+ prostate CSCs do not have a strong response to TKI treatment and that their response is context dependent. The TKIs imatinib and sunitinib were developed for and have a higher specificity for other tyrosine kinases. The lack of CD117+ cell response may be one reason for the failures of these TKIs in clinical studies 82 . Further, for prostate cancer, TKIs were given to patients with castration-resistant metastatic disease, which may be too late for the treatments to show efficacy. However, cabozantinib treatment in CRPC patients demonstrated tumor reduction and smaller bone metastases 83 . Cabozantinib, which was not tested in this study, had a higher specificity for CD117 than either imatinib or sunitinib 21 . CD117 expressing osteosarcoma cells are resistant to doxorubicin 69 . CD117 mutations in GIST are responsible for resistance to TKI treatment. 14% of GIST patients are initially resistant to imatinib, and 50% develop resistance within two years of treatment. For most patients, sunitinib will then be used and effective unless one mutation, D816H/V, is present which is resistant to both TKIs. Imatinib works better on inactive CD117 and prevents activation, but does not bind to activated CD117 84 . Further, SCF in the bone microenvironment alters the efficacy of treatments, especially TKIs, which have off-target effects in bone 85 . TKIs induce osteonecrosis in the jaw even when combined with bisphosphonates 86 . One possible reason may be stimulation of osteoclast and osteoblast differentiation by SCF. Another confounding factor is that tumor microenvironment SCF induces imatinib resistance by competing for the binding site with a higher affinity for CD117 81 . Thus, the high levels of SCF in the bone microenvironment may prevent TKI treatment from working properly on CD117 expressing prostate cancer cells, the numbers of which increase after exposure to the bone marrow 31 . The failures of prior TKI research may be due to the testing of inhibitors in patients who had already developed bone metastases 82 . This may be too late to alter progression and TKIs could be more effective earlier in the metastatic process. Further, failures may be due to the low specificity of current TKIs for CD117 21 .
In combination with our prior data showing a CD117+ CTC population in advanced prostate cancer patients, the data from this study demonstrate that CD117 expression in primary tumors and on CTCs may distinguish advanced cancer patients likely to have more aggressive tumors leading to recurrence and metastasis. Further research is required to verify that CD117 expressing cells represent CSCs in patients and that these cells initiate bone metastases, which would require the development of new bone metastases models 87 . In summary, we demonstrate that the CD117 subpopulation of C4-2 prostate cancer cells represents CSCs driving progression, migration, and TKI resistance.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.