Oncogenic Invasion

URPP Fellow: Evelyn Lattmann

Malignant melanoma is the most aggressive skin cancer in the world.  And not only is it quite deadly upon metastasis, but the worldwide incidence of cutaneous malignant melanoma is increasing more rapidly than almost any other cancer type in fair-skinned populations. The majority of melanoma patients (i.e. 80%) are diagnosed at an early enough stage that surgery alone is largely successful. However, the prognosis is quite poor for patients with metastatic melanoma.
During C. elegans larval development, a specialized cell called the anchor cell (AC) invades the underlying epidermis formed by the vulval cells. AC invasion is a highly reproducible process, as it is always the same cell that invades at a specific site and at a predetermined time point. During AC invasion, two basal laminae (BL) between the uterus and epidermis must be breached, which involves two distinct mechanisms: 1) guidance of the AC towards the epidermis and 2) breaching of the BL separating the two compartments. AC guidance depends on a netrin signal produced by the ventral nerve cord (VNC) and an unknown guidance cue from the epidermis. BL breaching, on the other hand, requires the two transcription factors FOS-1 (the ortholog of the human FOS oncogene), and EGL-43 (the homolog of the EVI1 oncogene. These two transcription factors induce in the AC the expression of several invasion effectors, such as the ZMP-1 metalloprotease, or the CDH-3 protocadherin. Finally, studies of AC invasion have not only identified genes that induce cell invasion but also identified genes that actively repress invasion to prevent ectopic BL breaching.
During the first phase of this URPP, we carried out an RNA interference screen in C. elegans to identify genes associated with invasive melanoma that may also be functionally involved in developmental cell invasion. For this purpose, we used anchor cell invasion in C. elegans as an in vivo invasion assay. Through this approach we identified novel pro-invasive genes that have so far not been associated with increased tumor cell invasion. Among the genes we further characterized are specific G1 cell cycle regulators that are frequently genetically altered by mutations and amplifications in melanoma, and components of the protein sumoylation pathway as well as specific sumoylation targets. In addition, some of the genes up-regulated in invasive melanoma cells have already been known to be involved in anchor cell invasion in the worm (e.g. AP-1 transcription factors, or the RAC GEF TRIO). Thus, we have identified among a long list of genes showing elevated expression in invasive melanoma a much shorter list of novel candidate genes that may be functionally involved in regulating melanoma cell invasion. Moreover, we have established 4D imaging of C. elegans larvae expressing different fluorescent reporters (e.g. for cell polarity or basement membrane breaching) in order to observe cell invasion in vivo and characterize the selected candidate genes in gain- and loss-of-function experiments in real time with high precision. To quantitate cell invasion in human cells, we have established XCelligence invasion assays coupled to growth factor stimulation to test the invasive potential of candidate genes in cancer cells including patient-derived melanoma cells. With this assay, we are testing the candidate genes identified in the C. elegans invasion screen described above for their roles in melanoma cell invasion.

Studying invading cancer cells in humans has obvious challenges; however, tumors shed cancer cells into
the bloodstream already early during disease. These circulating tumor cells (CTCs) may therefore be a promising, non-invasive means to get insights into a patient’s tumor. Yet, there is still controversy in how these CTCs relate to tumor progression. This mainly stems from the difficulty of isolation (e.g. different use
of markers, false positives) and/or lack of molecular characterization of individual CTCs due to limited material. Combining our expertise on melanoma biology, access to patient biopsies, and the label-free method of isolating CTCs from blood that is used in the group of Prof. Lars Steinmetz (Stanford University), bears great promise to tackle this challenge. Thus, the main goal of the proposed project is to isolate CTCs
by magnetic levitation and to determine the transcriptome of individual CTCs by single-cell sequencing using 10x Genomics. Ultimately, we strive to better understand the biology of CTCs in order to discover novel biomarkers and assess treatment efficacies (e.g. BRAF treatment) for adequate selection of melanoma therapy. Concretely, we will focus on stage IV melanoma patients that received treatment
with MAPK-inhibitors. CTCs will be isolated from peripheral blood mononuclear cells (PBMCs) at time point t0 (treatment begin) and at time point t1 (first follow-up at 3 months) from 10 patients. In order to determine the nature of these CTCs, single cell sequencing will be performed. The identity of melanoma CTCs will be evaluated by expression of melanoma specific markers (e.g. Tyrosinase, Melan-A/Mart-1, gp100, MITF, MAGE-A3) and CTC specific (e.g. EpCAM) genes. To obtain insight into the biology of CTCs by individual transcriptome analysis we will pursue the following five objectives (1) isolation of additional markers for CTCs (2) definition of transcriptional states of CTCs (3) molecular consequences (4) comparison with matched primary and secondary tumors (5) correlation with frequent melanoma-specific gene alterations. By fulfilling these objectives, we expect to obtain a molecular blueprint of individual CTCs of melanoma/cancer patients. This will be a crucial stepping-stone towards using CTCs as prognostic markers of melanoma progression and guides for therapeutic strategies.

Aims

  1. Isolation of additional markers for CTCs: To date there is no universal and exclusive CTC marker. EpCAM is the most commonly used CTC marker, however many CTCs lack this marker (e.g. in response to epithelial-to-mesenchymal transition (EMT), or CTCs of mesenchymal origin). Label-free isolation of CTCs will allow the detection of novel CTC markers.
  2. Definition of transcriptional states of CTCs: Four different transcriptional states have been described in PDX mouse models derived from BRAF treated melanoma patients (quiescent neural crest stem cell (NCSC), CD36+ ‘starved”-like melanoma cells (SMCs), MITFlow/SOX10low/AXLhigh mesenchymallike invasive cells, and MITFhigh ‘pigmented” cells). NCSC have been shown to be key drivers of resistance. Transcriptome analysis will allow for the determination of whether similar or distinct transcriptional states can be detected in CTCs.
  3. Molecular consequences: Based on the transcriptome data, markers for different CTC subtypes may be derived, which will allow their isolation by multiparametric FACS sorting. This will enable further downstream characterization (e.g. protein regulation, morphology and functional experiments such as RTCA xCELLigence and bioengineered skin assays).
  4. Comparison to matched primary and secondary tumors: Is the transcriptional landscape of CTCs related to the patient’s cancer? In order to answer this question, we will compare the transcriptome of the CTCs with single-cell sequencing of matched primary and secondary tumors.
  5. Correlation with frequent melanoma-specific gene alterations: Changes in gene expression may arise from epigenetic reprogramming and/or acquisition of mutations. With single-cell PCR we will assess mutations or alterations of candidate genes (e.g. genes from the “MELArray” that is tailored to the detection of frequent alterations in melanoma).

In summary, we have not only identified conserved regulators of cell invasion but also created a unique infrastructure to target melanoma invasion. Based on these very encouraging achievements, we will focus in the following period on further elucidating how these newly identified invasion regulators are integrated in invasive signaling pathways in human melanoma and perform in parallel a detailed functional characterization in C. elegans.