Trafficking of EGF family ligands in polarized epithelial cells
“Loss of polarity” is one of the first perturbations in the transformation process. Vectorial targeting of cargos (lipids/proteins) is a key component that governs establishment and maintenance of a polarized epithelium. Apico-basolateral trafficking and membrane identity is one aspect of polarity that we are studying using higher dimensional cultures to recapitulate the subtle trafficking phenotypes that are not appreciated in 2D plastic cultures. Moreover, knowledge acquired from these studies may be applicable to other trafficking problems where subcellular compartmentalization is key to cellular function e.g. nervous system, polycystic kidney diseases.
A major focus within this area is to understand the loss of epithelial polarity and associated transformation induced by loss of polarized trafficking of the EGFR ligand, epiregulin. Other EGF-like ligands currently being studied are neuregulins 1-4, and HBEGF.
Methods
Epithelial cells typically do not polarize with separate apical and basolateral surfaces when grown on glass or plastic. For our studies, we grow select epithelial cells on permeable support, like perforated polycarbonate/polystyrene filters. It allows for selective access to, manipulation of, and harvest from apical and basolateral surfaces.
Apical mistrafficking of EREG causes transformation.
Y156A substitution in the cytoplasmic domain of EREG leads to apical mislocalization in polarized MDCK cells in Transwell cultures. In nude mice subcutaneous xenografts, this EREG mutation leads to transformation and invasion of MDCK cells. In MatrigelTM cultures, apical EREG-expressing MDCK cells show aberrant cyst morphology with luminal growth of cells (arrow) and ectopic lumens (arrowhead), in addition to large central lumens.
Another method we employ in the lab for these studies is 3D Matrigel culture models. When allowed to grow suspended in gelatinous extracellular matrix (ECM) components (collagen or Matrigel), some epithelial cell lines form unilamellar spherical structures, termed cysts or acini. These structures show a greater resemblance to in vivo epithelia, as their basal membranes are in contact with the ECM and the apical surface contacts a de novo–generated apical lumen. In many ways, cysts may be considered the basic unit of the epithelium in vitro. In addition to the structural specializations seen in Transwell cultures, these ECM cultures (often termed 3D cultures) recapitulate features of epithelia, such as branching morphogenesis, and certain aspects of cellular transformation.
Role of receptor tyrosine kinase signaling in colorectal cancer and resistance to EGFR-targeted therapies
Cooperation between EGFR and other RTKs in cetuximab resistance. We hypothesize that multiple RTKs promote resistance to EGFR-directed therapies (e.g. cetuximab) thereby maintaining activation of shared downstream pro-oncogenic signaling pathways, like MAPK signaling. Inhibition of relevant RTKs (e.g. MET/RON) by targeted inhibitors (e.g. crizotinib) may help overcome CRC resistance to EGFR-directed therapies. Apart from establishing mechanisms of RTK crosstalk, translational efforts being pursued are highlighted in red. For example, combining cetuximab with crizotinib, cabozantinib, and ligand protease inhibitors. We are also developing expression-based and PET-based predictive biomarker for the effectiveness of cetuximab/crizotinib combination.
Colorectal cancer (CRC) remains the second leading cause of cancer-related deaths in the United States. Individuals with surgically unresectable stage 4 CRC have a five-year overall survival of 13%. Targeting of the receptor tyrosine kinase (RTK), EGF receptor (EGFR) with therapeutic monoclonal antibodies (cetuximab, panitumumab) is approved for use in wild-type KRAS advanced CRC. However, these antibodies as a single agent have response rates of 12-17% and expected progression-free survivals of only 3-4 months. Resistance may be present at the outset (de novo resistance) or develop during treatment (acquired resistance). Thus, there is a pressing need 1) to identify individuals most likely to respond (or not respond) to cetuximab and 2) to devise treatment strategies that would enhance response to cetuximab or prevent/delay cetuximab resistance. RAS/RAF mutations account for the majority of CRC cetuximab resistance, and individuals exhibiting these genetic modes of resistance are mostly excluded from receiving cetuximab or panitumumab. Non-genetic modes of cetuximab resistance, however, remain poorly defined. We have shown a new non-genetic mode of cetuximab resistance, due to activation of WT, non-amplified RTKs (MET/RON) as determined by their tyrosine phosphorylation (pMET/pRON) status.
We identified elevated pMET/pRON levels in the absence of their mutation or amplification as a cause of both de novo and acquired forms of cetuximab resistance using CRC cell lines grown in three-dimensional (3D) type-1 collagen. Moreover, addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity. We thus hypothesize that non-genetic modes of RTK activation are functionally relevant in CRC and individualized targeting of RTK signaling (e.g. MET/RON) may overcome CRC cetuximab resistance. Major focus of these studies are:
-
Determine the contribution of RTKs in CRC: role of RTK crosstalk in CRC progression and cetuximab resistance.
-
Assess the impact of disrupting RTK cooperation in CRC cetuximab resistance: disrupt RTK signaling axes by targeting relevant ligands and receptors using small molecule inhibitors or neutralizing antibodies in vitro and in vivo.
-
Devise rational drug combinations and strategies to identify subset of CRCs responding to MET/RON inhibition: generate expression-based and PET-based signatures that predict response to targeted therapies and their combinations.
Methods
CRC cell lines and patient-derived organoids (PDOs) are tested primarily in 3D (collagen, Matrigel). As needed, genetic and chemical manipulation of cells is performed and then tested in in vitro 3D cultures and in vivo in xenograft models. Depending on project needs, we also generate and analyze high-dimensional data (e.g. immunofluorescence, microscopy, RNA-seq, WES) combined with appropriate statistical analyses.