Background
Prostate cancer is a common malignancy in men, and can be treated successfully if the cancer is of low grade. Low-grade tumors are well-differentiated with glandular formation (adenocarcinoma). High-grade tumors are poorly differentiated with no glandular formation (non-adenocarcinoma). Androgen deprivation therapy can be effective when the cancer recurs after initial treatment. However, in many patients undergoing this treatment, the cancer becomes castration resistant. One notable tumor type in these advanced diseases is small cell carcinoma. It is highly aggressive, and does not respond well to anti-cancer agents [
1].
How do small cell carcinoma arise? How do cancer cells transition from a well-differentiated morphology to a poorly differentiated one? Relevant to answering these questions is the characterization of prostate cancer cells as either luminal-like (i.e., similar to normal luminal cells with a few hundred differentially expressed genes) or stem-like (i.e., dissimilar to luminal cells with thousands of differentially expressed genes) [
2,
3]. The former included mainly adenocarcinoma while the latter non-adenocarcinoma and small cell carcinoma. This dichotomy of cancer cell types was visualized in a principal components analysis (PCA) space generated from the transcriptomes of prostate luminal, basal, stromal, endothelial (differentiated cell types), plus those of stem cells [embryonic stem (ES), embryonal carcinoma (EC), induced pluripotent stem (iPS)] [
4]. Since stem cells give rise to somatic cells through differentiation, cancer cell differentiation might also be involved in the generation of multiple cancer cell types. Cancer cell differentiation could proceed from a cancer stem-like cell type to luminal-like adenocarcinoma. This differentiation could be arrested at intermediate stages to produce more stem-like types such as non-adenocarcinoma and small cell carcinoma. Alternatively, stem-like types could arise from de-differentiation as seen in reprogramming of differentiated somatic cells via forced expression of a set of stem cell transcription factors (scTF) [
5]. Other researchers suggested that transformation of basal epithelial cells, present in benign glands but not in tumor glands, gave rise to poorly differentiated cancer cells. CD44
+ CD49f
+ basal cells were postulated to be the prostate progenitor cells. Transformed basal cells (through in vitro transfection of vectors containing oncogenes) produced highly aggressive cancer cells [
6,
7]. However, transcriptomes of prostate cancer cell types analyzed, to date, evinced no expression signature of basal cells [
2,
8]. Basal cells express few, if any, stem cell markers. Rather, they represent a differentiated cell type as shown by the different gene expression of basal cells in the prostate and bladder [
8,
9].
Previously, we reported the presence of scTF LIN28A, NANOG, POU5F1 and SOX2 in a small cell carcinoma patient-derived xenograft (PDX) line LuCaP 145.1 but absent in adenocarcinoma PDX lines [
10]. We chose these four scTF specifically because they can perform reprogramming [
11]. In addition, LuCaP 145.1 was found to share expression of many genes with stem cells, including the down-regulation of β2-microglobulin (B2M) [
10]. B2M is a so-called housekeeping marker in adult cell types, both normal and cancerous. It is commonly employed as a control in reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression. More recently established LuCaP including non-adenocarcinoma and small cell carcinoma allowed us to examine scTF expression in lines other than LuCaP 145.1. The > 30 LuCaP lines were established from human tumors and propagated in male SCID mice. Both transcriptomic analysis and immunostaining have shown concordance between LuCaP tumor cells and their corresponding human donor tumor tissues [
12‐
16]. Significantly, we also wanted to determine if the scTF genes in LuCaP 145.1 were responsible for the gene expression of stem-like cancer types. Accordingly, these genes were cloned from LuCaP 145.1 into expression vectors for cell transfection. The goal of this research was to test the hypothesis that adenocarcinoma vs. non-adenocarcinoma/small cell carcinoma are related through cancer de-differentiation.
Discussion
Small cell carcinoma is a rare but lethal form of prostate cancer comprising 5% of cancers [
25]. The presence of prostate cancer-specific TMPRSS2-ERG fusion in both adenocarcinoma and small cell carcinoma of the same tumor cases suggests a direct lineage [
26]. In one way, prostate small cell carcinoma with neuroendocrine differentiation is regarded as trans-differentiation of adenocarcinoma based on research using LNCaP [
27]. In our research, the transcriptome of small cell carcinoma LuCaP 145.1 was found to be closest to that of ES than those of other prostate cancer cell types - cell lines, cells isolated from primary tumors, and adenocarcinoma LuCaP lines. The ES proximity was confirmed by the expression of LIN28A, NANOG, POU5F1 and SOX2 in LuCaP 145.1. The newly available LuCaP 93 small cell carcinoma was found to express these scTF, but with lower NANOG. The strength of NANOG expression could affect the conversion of cancer cell density from [epi] to [strom]. LuCaP 145.1, as indicated by banding at [strom], had completely lost its epithelial characteristics (i.e., like stem cells). The other tumors still contained cells banding at both [epi] and [strom]. The difference in gene expression among the LuCaP small cell carcinoma lines reflected the finding of multiple small cell carcinoma subtypes in human tumors [
28]. Expression heterogeneity was also found among LuCaP adenocarcinoma lines regarding the scTF genes – many with POU5F1, a few with LIN28A, none with SOX2 and NANOG [
2,
10]. The aggressive behavior and therapy resistance of prostate small cell carcinoma could be attributed to their stem-likeness, because stem cells are equipped to survive over an organism’s lifespan. Reports in the literature have documented the association between LIN28 [
29], NANOG [
30], POU5F1 [
31], and SOX2 [
32] and prostate cancer aggressiveness individually.
Our experiments also tried to determine if the scTF in LuCaP 145.1 were responsible for its stem-like expression. These genes were cloned for reprogramming testing. Transfection of 293F fibroblasts as well as prostate cancer cells LNCaP, C4–2B, and PC3 produced cells with stem-like colony morphology and down-regulated B2M, indicating that the proteins encoded by these genes were functional. Their functionality is equivalent to that of the same scTF cloned from ES cells being used to reprogram somatic cells [
11], prostate cancer-associated stromal cells [
17], and LuCaP adenocarcinoma lines [
10]. The transformed cells could be propagated in serum-free media under hypoxia, which could inhibit cells like parental LNCaP [
33]. Furthermore, increase in B2M expression is associated with differentiation while decrease with de-differentiation as exhibited by LuCaP 145.1.
We postulate that the transition of prostate cancer from adenocarcinoma to non-adenocarcinoma and small cell carcinoma involves activation of scTF genes in the sequence of POU5F1 → LIN28A/SOX2 → NANOG with tumor cells adopting a more de-differentiated state. SOX2, for example, is found in the undifferentiated developing prostate [
32], and is responsible for the maintenance of neural progenitors [
34]. Our proposed scheme of prostate cancer de-differentiation could proceed from Gleason pattern 3 adenocarcinoma/LNCaP → C4–2B → Gleason pattern 4 → PC3/non-adenocarcinoma (LuCaP 173)/SOX2
+ small cell carcinoma LuCaP 49 [
2] → LuCaP 93 → LuCaP 145. It would be interesting to compare prostate small cell carcinoma with small cell carcinoma of lung and bladder, which have recently been analyzed by exome sequencing [
35]. The exome sequencing data revealed no single thematic pattern for these small cell carcinoma such as a high number of DNA mutations, which is similar to what was found by exome sequencing of LuCaP small cell carcinoma and LuCaP adenocarcinoma [
12]. The poorly differentiated prostate small cell carcinoma phenotype is, at least, not due to an accumulation of genomic changes over time. We could explore whether the four scTF are expressed by these other small cell carcinoma types, whether reprogrammed non-small cell lung or urothelial cancer cells show similar expression as reprogrammed prostate cancer cells. We hypothesize that stem-like prostate cancer cells may also respond to stromal cell signaling as shown by the germ cell tumor-derived NCCIT.
The advantage afforded by our plasmid vectors includes biosafety over the previously used lentiviral vectors, especially since these scTF genes could be potentially oncogenic (
http://cancer.sanger.ac.uk/cosmic/census/tables?name=symbol) due to their expression in cancer. Ample plasmid DNA could be obtained from small cultures while lentiviral vectors require expertise, high cost and a complicated procedure to produce transfection-ready stocks [
18]. Other commercially available viral vectors, e.g., CytoTune Sendai virus [
36], are also expensive for relatively small amounts of DNA. We have also tried other plasmid-based vectors [
37] in our earlier studies, but found very low transformation efficiency due perhaps to the need for co-transfection of several separate plasmids. The drug selection marker (neo) allows transformation of fast growing cancer cell lines in which untransfected cells would otherwise overwhelm transfected cells without it. Additionally, vectors containing antisense scTF genes (cloned in the 3′ → 5′ orientation) can be used to inactivate the genes in LuCaP 145.1, for example, to determine if forced differentiation could lead to an adenocarcinoma-like derivative.
Acknowledgments
We thank Holly Nguyen of Urology in harvesting and donating the LuCaP xenografts; Christopher Cavanaugh of UW ISCRM Stem Cell Core for advice and assistance; Pamela Troisch of the Institute for Systems Biology, Sengkeo Srinouanprachanh and Theo Bammler of Functional Genomics UW-DEOHS for array analysis and chip scanning, respectively.