Modulating Cancer Stem Cell Signaling in Thoracic Malignancies

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

During the past year, we have continued our efforts to explore the potential utility of induced pluripotent stem cell (iPSC) models for characterizing epigenetic mechanisms mediating sternness, treatment resistance, and metastatic potential of thoracic malignancies to identify novel therapeutic targets in these neoplasms. Briefly, we have extended our studies related to the mechanisms by which Additional Sex Combs Like-3 (ASXL3) promotes pluripotency in normal stem cells and enhances epigenetic plasticity in thoracic cancers. In collaboration with Dr. Mirit Aladjem, we observed that ASXL3 is upregulated as cells transit from G1 to early S phase and enhances firing at origins of DNA replication while decreasing speed of DNA strand synthesis resulting in reversal of early vs late gene synthesis. In additional experiments, tamoxifen- inducible knockout of ASXL3 diminished efficiency of reprogramming of normal cells to iPSC and impaired clonogenic growth of these cells. siRNA-mediated knockdown of ASXL3 markedly reduced bZIP transcription factor occupancy on chromatin resulting in significant toxicity in cultured human SCLC lines. We established a novel SCLC GEMM and observed that CRE-mediated knockout of ASXL3 "locked" SCLC in an ASCL1 phenotype and markedly extended survival of mice bearing inducible and highly lethal RPM cancers. Experiments are in progress to characterize the epigenomic and immunologic phenotypes of A-RPM vs RPM SCLC and to correlate the murine model findings with human SCLC. An EIR pertaining to this novel GEMM is being finalized for CCR review. We have previously reported that upregulation of ASXL3 in SCLC correlates with increased gene copy number. We have extended these experiments and observed that overexpression of ASXL3 appears to be associated with translocation of ASXL3 and nearby genes on chromosome 18 to, as yet, unidentified loci on chromosomes 5 and 10. FISH experiments are underway to further examine the frequency and site(s) of ASXL3 translocations in human SCLC lines and tumor specimens as such events could generate novel fusion proteins that can be monitored and possibly targeted in the clinic. A comprehensive manuscript describing these findings is in the final stages of preparation for peer review. Whereas cancer stem cells (CSCs) have been implicated in treatment resistance, metastatic potential, and lethality of human malignancies, efforts to study and therapeutically target CSCs have been hampered by the lack of a consistent marker of pluripotency - even among cancers of similar histology. Because ASXL3 appears to be an epigenetic driver in SCLC which typically exhibit high plasticity and stemness, we used RNA-seq, qRT-PCR, and immunoblot techniques to examine expression levels of ASXL3 and stem cell-related genes in SCLC, non-small cell lung cancer (NSCLC), esophageal cancer, and pleural mesothelioma lines (n= 17) cultured either under standard 2-D or non-adherent (3-D; spheroid) conditions, as well as primary thoracic malignancies (n=7) and pulmonary metastasectomy specimens (n= 10) cultured as spheroids, and induced pluripotent stem cells (iPSC) established from normal lung (Lu) or esophageal (Eso) epithelial cells, or pleural mesothelial (Meso) cells. Interestingly, we observed that similar to Lu-iPSC, ASXL3 was upregulated in Eso- and Meso-iPSC. ASXL3 expression peaked during a transient stage of epigenetic reprogramming and diminished as cells became committed to either prime or naïve iPSC states. In contrast to typical Lu-, Eso-, or Meso-iPSC that formed mature teratomas in NSG mice, rare iPSC lines in which ASXL3 expression remained markedly elevated formed high-grade embryonal carcinomas. Although undetectable in established cancer lines or surgical specimens cultured under 2-D conditions, ASXL3 expression was enriched in spheroids isolated from cancer lines as well as primary thoracic tumors and pulmonary metastases cultured on Matrigel. ASXL3+ cell populations from cell lines or tumor specimens co-expressed CD133 and ALDH1, and to a lesser extent NANOG, and exhibited elevated stemness scores, in-vitro chemoresistance, and enhanced tumorigenicity and metastatic potential in immunodeficient mice. Knockdown of ASXL3 decreased stemness scores and downregulated a variety of pathways involved in stem cell maintenance including E2F targets, Pathways in Cancer, Aurora B signaling, and G2M checkpoint. Collectively, these studies indicate that ASXL3 is a novel marker and potential mediator of stemness in primary as well as secondary thoracic cancers. The aforementioned studies pertaining to ASXL3 have been hampered by the lack of high-specificity antibodies and our inability to overexpress full length ASXL3 in any cell regardless of endogenous ASXL3 expression. Nevertheless, we continue this line of research because such efforts could provide novel insights into the role of ASXL3 during the development of highly lethal SCLC and establish ASXL3 as a novel target for eradication of cancer stem cells regardless of tumor histology.