This week we profile a recent publication in Science Advances from the Sorensen Lab. First author Hai-Feng Zhang (pictured, left) and senior author Poul Sorensen (right) share more details about what went into this publication, why these research questions excite them, and the future direction of their work and the lab.
Can you provide a brief overview of your lab’s current research focus?
A major interest in my laboratory is to identify and understand how the tumor microenvironment imparts potentially lethal stresses on childhood cancer cells, including oxidative stress, hypoxia, and nutrient deprivation. Tumor cells must acutely adapt to these stresses to survive, potentially leading to emergence of aggressive clones with high metastatic capacity. By understanding these processes, we can design therapeutic strategies to attempt to reduce metastatic competence in childhood cancers. This is an urgent clinical unmet need, as relapsed and metastatic childhood cancers have dismal survival rates which have not improved for decades. We also use immunotherapy approaches for targeting high-risk childhood cancers. A major roadblock is the paucity of known tumor-associated surface proteins that could potentially serve as targets for immunotherapy in these diseases. We have therefore developed a world-class pipeline for the discovery of such targets in childhood malignancies, using plasma membrane enrichment linked to mass spectrometry based proteomics to identify novel surface targets.
What is the significance of the findings in this publication?
Genomic amplification of the MYCN gene on chromosome 2 (designated MNA+) is a defining feature of high-risk neuroblastoma (NB), and predicts poor prognosis in these aggressive childhood cancers. It has been largely assumed that the MYCN oncogene is the sole genetic driver of MYCN-amplified NB. For the first time, we uncovered that GREB1 (Growth Regulating Estrogen receptor Binding 1), not in the MYCN amplicon but potentially activated by being adjacent to the amplicon, is frequently co-expressed with MYCN, and is like a partner in crime with MYCN in this highly aggressive NB subtype.
Specifically, although not widely appreciated, genes neighboring the MYCN locus are frequently coamplified with MYCN in MNA+ NB. However, whether these genes contribute to MNA+ NB pathogenicity and aggressiveness independently of MYCN or are merely passengers in the oncogenic process remains largely unknown. Among those genes, GREB1 encodes a transcription factor known to mainly promote tumorigenicity in various hormone-dependent cancers, including breast, ovarian, prostate, and endometrial cancers. We discovered that this hormone-related factor GREB1 surprisingly plays a crucial role in MNA+ NB, a type of cancer mainly found in infants and children under 5 years old.
Moreover, through integrated transcriptomic analysis, we have identified a GREB1-controlled gene signature in MNA+ NB independent of MYCN regulation. Among this novel GREB1-gene signature, we identified that Myosin 1B (MYO1B), an unconventional myosin that regulates intracellular protein trafficking, mediates global secretome reprogramming. In the secretome of MNA+NB cells, we found that a cytokine called MIF is highly co-localized with MYO1B-positive budding vesicles on cell surface, and MYO1B directly promotes MIF secretion. These processes contributes to MNA+ NB aggressiveness, including tumor cells invasiveness and metastatic capacity as revealed by a chick embryo metastasis model. Therefore, we have defined a previously unrecognized GREB1-MYO1B-MIF axis that contributes to the pathobiology of MNA+ NB, but in a manner that is independent of MYCN.
What kind of impact do you hope your research will have?
Given the challenges in directly targeting MYCN pharmacologically, a notorious “undruggable” oncoprotein, our new findings may inform the field to recognize these new potential therapeutic targets for patients with high-risk MNA+ NB, such as GREB1, MYO1B and MIF.
Where do you see this research going in the next five/ten years?
Despite the well-known oncogenic driver role of MYCN in MNA+ NB, our findings suggest that the “partner in crime” GREB1 is also crucial for MNA+ NB cell growth and survival. In the next five/ten years, the attention of this specific research field may start to also cover this class of so-called “passenger genes” that turn out to be functionally significant, and may be co-targeted with driver oncogenes, thereby to enhance therapeutic efficacy and prevent tumor relapse.
Was there a new process (assay or data analysis) that you employed during this study? If not, is there something on your radar that you want to try?
An uncommonly used strategy called pSILAC-Click (i.e. pulsed stable isotope labeling with amino acids in cell culture, or pSILAC, combined with Click chemistry) was used in our study to identify the global secretome in cancer cells. This approach allows specific labeling and purification of cell-derived proteins in conditioned medium, while minimizing the immense background noise impose by large amounts of albumin proteins in the conditioned medium. Thus, this approach enables analysis of the acute secretome in cells cultured under normal growth medium instead of serum starvation conditions that are required by more conventional approaches, overcoming interference by existing proteins in the culture media.
What new questions are you really excited about right now?
The proto-oncogenic function for GREB1 remains relatively understudied. Our study further highlights the potential oncogenic role of GREB1 in distinct cancers, in particular a non-hormone-related cancer such as pediatric NB. Indeed, in support of this notion, various fusion genes involving GREB1 have been identified in cancers, including GREB1-NCOA1, GREB1-NCOA2, GREB1-NR4A3, GREB1-SS18, and ESR1-GREB1, mainly in uterine tumors resembling ovarian sex-cord tumor (UTROSCT). The exact role of these potentially oncogenic fusions involving GREB1 remains unclear during tumorigenesis.
Moreover, our transcriptomic analysis revealed potential GREB1 regulation of biological processes such as chromosome organization, DNA damage repair, and axonogenesis. Furthermore, the top gene categories suppressed by GREB1 included circadian regulation of gene expression, the dysregulation of which affects various hallmarks of cancer. These previously unknown findings warrant future in-depth studies.
Funding for this study was provided by: the NIH U54 Pediatric Immunotherapy Discovery and Development Network, and a St. Baldrick’s Foundation/AACR/SU2C Pediatric Dream Team Translational Research Grant (to P.H.S.). This work was also supported by funds from the BC Cancer Foundation (to P.H.S.) and funds from Terry Fox Research Institute Team Grant 1021 (to P.H.S.). H.-F.Z. is funded by a fellowship from the Canadian Institutes of Health Research (CIHR) and a trainee award from the Michael Smith Foundation for Health Research partnered with the Lotte and John Hecht Memorial Foundation.
