Metastasis is a multistep process requiring cancer cell signaling, invasion, migration, survival and proliferation. These processes require dynamic modulation of cell surface proteins by endocytosis. Given this functional connection, it has been suggested that endocytosis is dysregulated in cancer. To test this, we developed In-Cell ELISA assays to measure three different endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis and clathrin-independent endocytosis and compared these activities using two different syngeneic models for normal and oncogene-transformed human lung epithelial cells. We found that all endocytic activities were reduced in the transformed vs. normal counterparts. However, when we screened 29 independently isolated non-small lung cancer (NSCLC) cell lines to determine whether these changes were systematic, we observed significant heterogeneity. Nonetheless, using hierarchical clustering based on their combined endocytic properties, we identified two phenotypically distinct clusters of NSCLCs. One co-clustered with mutations in KRAS, a mesenchymal phenotype, increased invasion through collagen and decreased growth in soft agar; whereas the second was enriched in cells with an epithelial phenotype. Interestingly, the two clusters also differed significantly in clathrin-independent internalization and surface expression of CD44 and CD59. Taken together, our results suggest that endocytotic alternations in cancer cells that affect cell surface expression of critical molecules have a significant influence on cancer-relevant phenotypes, with potential implications for interventions to control cancer by modulating endocytic dynamics

The fibroblast growth factor receptor FGFR1 is a therapeutic target under investigation in multiple solid tumors and clinical trials of selective tyrosine kinase inhibitors (TKI) are underway. Treatment with single TKI represents a logical step towards personalized cancer therapy, but intrinsic and acquired resistance mechanisms limit their long-term benefit. In this study, we deployed RNAi-based functional genomic screens to identify protein kinases controlling the intrinsic sensitivity of FGFR1-dependent lung cancer and head and neck squamous cell cancer (HNSCC) cells to ponatinib, a multi-kinase FGFR-active inhibitor. We identified and validated a synthetic lethal interaction between Mammalian Target of Rapamycin (MTOR) and ponatinib in non-small cell lung carcinoma cells. Additionally, treatment with MTOR-targeting shRNAs and pharmacological inhibitors revealed that MTOR is an essential protein kinase in other FGFR1-expressing cancer cells. The combination of FGFR inhibitors and MTOR or AKT inhibitors resulted in synergistic growth suppression vitro. Notably, tumor xenografts generated from FGFR1-dependent lung cancer cells exhibited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547, but when combined with the MTOR inhibitor, AZD2014, significantly attenuated tumor growth and prolonged survival. Our findings support the existence of a signaling network wherein FGFR1-driven ERK and activated MTOR/AKT represent distinct arms required to induce full transformation. Further, they suggest clinical efficacy of treatments for FGFR1-driven lung cancers and HNSCC may be achieved by combining MTOR inhibitors and FGFR-specific TKI.

Survival rates associated with pancreatic cancer remain dismal despite advancements in detection and treatment strategies. Genetically engineered mouse models of pancreatic tumorigenesis have gained considerable attention based on their ability to recapitulate key clinical features of human disease including chemotherapeutic resistance and fibrosis. However, it is unclear if transgenic systems exemplified by the KrasG12D/Trp53R172H/Pdx-1-Cre (KPC) mouse model recapitulate the functional heterogeneity of human pancreatic tumors harboring distinct cells with tumorigenic properties. To facilitate tracking of heterogeneous tumor cell populations, we incorporated a luciferase-based tag into the genetic background of the KPC mouse model. We isolated pancreatic cancer cells from multiple independent tumor lines and found that roughly 1 out of 87 cells exhibited tumorigenic capability. Notably, this frequency is significantly higher than reported for human pancreatic adenocarcinomas. Cancer stem cell (CSC) markers including CD133, CD24, Sca-1, and functional Aldefluor activity were unable to discriminate tumorigenic from nontumorigenic cells in syngeneic transplants. Furthermore, three-dimensional spheroid cultures originating from KPC tumors did not enrich for cells with stem-like characteristics, and were not significantly more tumorigenic than cells cultured as monolayers. Additionally, we did not observe significant differences in response to gemcitabine or salinomycin in several isolated sub-populations. Taken together, these studies show that the hierarchical organization of CSCs in human disease is not recapitulated in a commonly used mouse model of pancreatic cancer, and therefore provide a new view of the phenotypic and functional heterogeneity of tumor cells.

Disease relapse in cancer patients many years after clinical remission, often referred to as cancer dormancy, is well documented but remains an incompletely understood phenomenon on the biologic level. Recent reviews have summarized potential models that can explain this phenomenon, including angiogenic, immunologic, and cellular dormancy. We focus on mechanisms of cellular dormancy as newer biologic insights have enabled better understanding of this process. We provide a historical context, synthesize current advances in the field, and propose a mechanistic framework that treats cancer cell dormancy as a dynamic cell state conferring a fitness advantage to an evolving malignancy under stress. Cellular dormancy appears to be an active process that can be toggled through a variety of signaling mechanisms that ultimately downregulate the RAS/MAPK and PI(3)K/AKT pathways, an ability that is preserved even in cancers that constitutively depend on these pathways for their growth and survival. Just as unbridled proliferation is a key hallmark of cancer, the ability of cancer cells to become quiescent may be critical to evolving malignancies, with implications for understanding cancer initiation, progression, and treatment resistance. Cancer Res; 75(23); 1–9. ©2015 AACR.

Intratumoral heterogeneity helps drive the selection for diverse therapy-resistant cell populations. In this study, we demonstrate the co-existence of two therapy-resistant populations with distinct properties that are reproducibly enriched under conditions that characterize tumor pathophysiology. Breast cancer cells that survived chemotherapy or hypoxia were enriched for cells expressing the major hyaluronic acid receptor CD44. However, only CD44hi cells that survived chemotherapy exhibited cancer stem cell (CSC) phenotypes based on growth potential and gene expression signatures that represent oncogenic signaling and metastatic prowess. Strikingly, we identified TGF-β2 as a key growth promoter of CD44hi cells that survived chemotherapy but a growth inhibitor of cells that survived hypoxia. Expression of the TGFβ receptor TGFβR1 and its effector molecule SMAD4 was required for enrichment of CD44hi cells exposed to the chemotherapeutic drug epirubicin, which suggests a feed-forward loop to enrich for and enhance the function of surviving CSCs. Our results reveal context-dependent effects of TGF-β2 signaling in the same tumor at the same time. The emergence of distinct resistant tumor cell populations as a consequence of prior therapeutic intervention or microenvironmental cues has significant implications for the responsiveness of recurring tumors to therapy.