Razidlo, Gina L.

Abstract Oncogene-induced metabolic reprogramming is a hallmark of pancreatic cancer (PDAC), yet the metabolic drivers of metastasis are unclear. In PDAC, obesity and excess fatty acids accelerate tumor growth and increase metastasis. Here, we report that excess lipids, stored in organelles called lipid droplets (LD), are a key resource to fuel the energy-intensive process of metastasis. The oncogene KRAS controlled the storage and utilization of LD through regulation of hormone-sensitive lipase (HSL), which was downregulated in human PDAC. Disruption of the KRAS–HSL axis reduced lipid storage, reprogrammed tumor cell metabolism, and inhibited invasive migration in vitro and metastasis in vivo. Finally, microscopy-based metabolic analysis revealed that migratory cells selectively utilize oxidative metabolism during the process of migration to metabolize stored lipids and fuel invasive migration. Taken together, these results reveal a mechanism that can be targeted to attenuate PDAC metastasis. Significance: KRAS-dependent regulation of HSL biases cells towards lipid storage for subsequent utilization during invasion of pancreatic cancer cells, representing a potential target for therapeutic intervention. See related commentary by Man et al., p. 4886

Dyn2 and α-actinin 4 interact directly to regulate invasive cell migration and the stabilization of invadopodia. Intriguingly, this is specific for α-actinin 4, and not the highly related protein α-actinin 1. These findings elucidate a novel mechanism of regulating cell migration, with important implications for invasive tumor cells.

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of only 9%, due in part to its robust metastatic capability. Metastatic invasion is a particularly energy-intensive cellular process; thus, novel therapeutic strategies could include targeting the energy source for tumor cell invasion and migration. Excess lipids, present due to increased fatty acid synthesis or obesity, could provide a fuel source for cancer progression and metastasis. Neutral lipids are stored in organelles called lipid droplets (LDs). How LDs contribute to PDAC tumor cell invasion and metastasis remains unknown. We hypothesize that PDAC cells can store excess lipid to drive metastatic invasion. Indeed, introduction of excess exogenous fatty acids augments LD production and accumulation in PDAC cells and leads to enhanced PDAC cell motility and invasion in vitro. LDs are metabolized by the concerted action of lipases. Importantly, we have found that treatment of tumor cells with lipase inhibitors blocks tumor cell invasion, indicating that utilization of stored lipids via lipase-mediated LD breakdown is required for invasive migration. Hormone sensitive lipase (HSL) is a lipolytic enzyme, best characterized in adipose tissue, that is critical to the breakdown and utilization of LDs in vitro and in vivo. We have made the surprising observation that oncogenic KRas downregulates expression of HSL in pancreatic cancer cells, which shifts the cells towards lipid storage. In PDAC, mutational activation of the oncogene KRas occurs in 95% of tumors and contributes to metabolic rewiring of tumor cells. Our data suggest that the metabolic shift induced by mutant KRas is comprised of decreased LD utilization via downregulation of HSL. Indeed, re-expression of HSL shifts tumor cell metabolism towards elevated oxidative phosphorylation. Importantly, KRas-mutant cells still require the catalytic activity of HSL for tumor cell invasion, even though HSL expression is downregulated. Using redox ratio imaging, we have found that migratory cells selectively utilize oxidative phosphorylation during the process of migration to metabolize stored lipid droplets and fuel invasive migration. Finally, depletion of LD stores via overexpression of HSL blocks tumor cell invasion in vitro and metastasis in mice, even in the context of oncogenic KRas. These data indicate that regulation of HSL is important for both lipid storage and maintaining cancer cell invasiveness. Therefore, we propose a novel mechanism by which oncogenic KRas mediates HSL activity in a tunable manner, thereby altering the metabolism of PDAC cells and leading to increased metastatic ability. This abstract is also being presented as Poster B47. Citation Format: Cody Rozeveld, Ryan Schulze, Lizhi Zhang, Gina L Razidlo. KRas modulates pancreatic cancer cell metabolism and invasive potential through the lipase HSL [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR09.

The process by which tumor cells mechanically invade through surrounding stroma into peripheral tissues is an essential component of metastatic dissemination. The directed recruitment of the metalloproteinase MT1-MMP to invadopodia plays a critical role in this invasive process. Here, we provide mechanistic insight into MT1-MMP cytoplasmic tail binding protein 1 (MTCBP-1) with respect to invadopodia formation, matrix remodeling, and invasion by pancreatic tumor cells. MTCBP-1 localizes to invadopodia and interacts with MT1-MMP. We find that this interaction displaces MT1-MMP from invadopodia, thereby attenuating their number and function and reducing the capacity of tumor cells to degrade matrix. Further, we observe an inverse correlation between MTCBP-1 and MT1-MMP expression both in cultured cell lines and human pancreatic tumors. Consistently, MTCBP-1–expressing cells show decreased ability to invade in vitro and metastasize in vivo. These findings implicate MTCBP-1 as an inhibitor of the metastatic process.

Abstract Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of T-cell malignancies that generally demonstrate aggressive clinical behavior, often are refractory to standard therapy, and remain significantly understudied. The most common World Health Organization subtype is PTCL, not otherwise specified (NOS), essentially a “wastebasket” category because of inadequate understanding to assign cases to a more specific diagnostic entity. Identification of novel fusion genes has contributed significantly to improving the classification, biologic understanding, and therapeutic targeting of PTCLs. Here, we integrated mate-pair DNA and RNA next-generation sequencing to identify chromosomal rearrangements encoding expressed fusion transcripts in PTCL, NOS. Two of 11 cases had novel fusions involving VAV1, encoding a truncated form of the VAV1 guanine nucleotide exchange factor important in T-cell receptor signaling. Fluorescence in situ hybridization studies identified VAV1 rearrangements in 10 of 148 PTCLs (7%). These were observed exclusively in PTCL, NOS (11%) and anaplastic large cell lymphoma (11%). In vitro, ectopic expression of a VAV1 fusion promoted cell growth and migration in a RAC1-dependent manner. This growth was inhibited by azathioprine, a clinically available RAC1 inhibitor. We also identified novel kinase gene fusions, ITK-FER and IKZF2-ERBB4, as candidate therapeutic targets that show similarities to known recurrent oncogenic ITK-SYK fusions and ERBB4 transcript variants in PTCLs, respectively. Additional novel and potentially clinically relevant fusions also were discovered. Together, these findings identify VAV1 fusions as recurrent and targetable events in PTCLs and highlight the potential for clinical sequencing to guide individualized therapy approaches for this group of aggressive malignancies.

Abstract Pancreatic cancer, one of the most lethal forms of human cancer, is largely resistant to many conventional chemotherapeutic agents. Although many therapeutic approaches focus on tumor growth, metastasis is a primary factor contributing to lethality. Therefore, novel therapies to target metastatic invasion could prevent tumor spread and recurrence resulting from local and distant metastasis. The protein Vav1 is aberrantly expressed in more than half of pancreatic cancers. Its expression promotes activation of Rac and Cdc42 and leads to enhanced invasion and migration, as well as increased tumor cell survival and proliferation, suggesting that Vav1 could be a potent therapeutic target for pancreatic cancer. The purine analogue azathioprine, well known for its function as an anti-inflammatory compound, was recently shown to function by inhibiting Vav1 signaling in immune cells. We therefore hypothesized that azathioprine could also inhibit Vav1 in pancreatic tumor cells to reduce its proinvasive functions. Indeed, we have found that treatment of cultured pancreatic tumor cells with azathioprine inhibited Vav1-dependent invasive cell migration and matrix degradation, through inhibition of Rac and Cdc42 signaling. Furthermore, azathioprine treatment decreased metastasis in both xenograft and genetic mouse models of pancreatic cancer. Strikingly, metastasis was dramatically reduced in Vav1-expressing tumors arising from p48Cre/+, KrasG12D/+, p53F/+ mice. These inhibitory effects were mediated through Vav1, as Vav1-negative cell lines and tumors were largely resistant to azathioprine treatment. These findings demonstrate that azathioprine and related compounds could be potent antimetastatic agents for Vav1-positive pancreatic tumors. Cancer Res; 75(14); 2907–15. ©2015 AACR.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of human cancer, due in large part to a high incidence of metastasis and resistance to conventional chemotherapies. As such, new therapies are needed to halt the metastatic process. The proto-oncogene Vav1 is ectopically expressed in over half of human pancreatic cancers, and its expression correlates with a poor prognosis in humans and promotes survival and transformation in isolated tumor cells. We have found that Vav1, through its GEF activity towards Rac1, also potently induces migration by pancreatic tumor cells. In addition, Vav1 also signals through Cdc42 to drive the formation of invadopodia and matrix degradation, which also promotes tumor cell invasion. Importantly, the activation of Vav1 circumvents the requirement for active Src in this process. Because Vav1 is ectopically expressed in pancreatic cancers and is a potent regulator of tumor cell survival, proliferation, and invasive migration, it could provide a specific therapeutic target for these tumors. Recently, the anti-inflammatory agent azathioprine was described as an inhibitor of Vav1/Rac1 activity in hematopoietic cells. Therefore, we hypothesized that azathioprine could also inhibit the Vav1-driven processes of proliferation and invasion in pancreatic tumor cells. Indeed, we have determined that azathioprine treatment of cultured human PDAC cells reduces viability and proliferation. Further, similar to siRNA-mediated depletion of Vav1, azathioprine treatment reduces cell migration, as well as the matrix degradation required for invasion. Vav1-expressing tumor cells were more sensitive to azathioprine than Vav1-negative cells, supporting the premise that the inhibitory effects of azathioprine are mediated through Vav1. Finally, azathioprine also reduces metastasis in a xenograft model of pancreatic cancer. Taken together, these data uncover a role for aberrantly expressed Vav1 in regulation of the cytoskeletal machinery required for migration and invasion, and in the promotion and metastasis of pancreatic cancer. Even with the expression of multiple Rho family GEFs, tumor cells that upregulate Vav1 have become dependent upon this exchange factor, implicating Vav1 as a potent node for therapeutic intervention. Supported by R01 CA104125 and R03 CA155778 to M.A.M. Citation Format: Gina L. Razidlo, Christopher Magnine, Arthur C. Sletten, Rachel M. Hurley, Mark A. McNiven. Attenuation of pancreatic cancer cell migration and invasion through a targeted inhibition of the Rac GEF Vav1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4026. doi:10.1158/1538-7445.AM2014-4026

Abstract Metastatic invasion of tumors into peripheral tissues is known to rely upon protease-mediated degradation of the surrounding stroma. This remodeling process is dependent upon complex, actin-based specializations of the plasma membrane termed “invadopodia” that act to both sequester and release matrix metalloproteases. Here we report that cells of mesenchymal origin, including fibroblasts, pancreatic stellate cells, and tumor-associated fibroblasts, degrade substantial amounts of surrounding matrix by a mechanism independent of conventional invadopodia. First, as tumor cell-derived invadopodia are dependent upon the action of the GTPase Dyn2, loss of Dyn2 function inhibits invadopodia-based matrix degradation by pancreatic tumor cells. In surprising contrast, we have found that loss of Dyn2 function in mesenchymal cells does not inhibit degradation, but actually leads to a marked increase. Second, these degradative sites lack the punctate shape of conventional invadopodia but are instead spread along the cell base and are reticular and/or fibrous in character. Third, inhibition of either Src or Cdc42 function, known to drive invadopodia formation, had no effect on the mesenchymal matrix degradation induced by loss of Dyn2. Taken together, these data indicate that this mechanism of degradation is independent of classical invadopodial degradation. To define the mechanistic processes that differ between tumor and stromal cell types, we screened for matrix metalloprotease activity using zymography. An unexpected and significant upregulation of MMP2 activity in mesenchymal-derived cells upon loss of Dyn2 was observed, whereas MMP2 was nearly completely absent in the tumor cells. Further, loss of Dyn2 resulted in an upregulation of surface-bound MT1-MMP, known to activate MMP2. In contrast, loss of Dyn2 in the pancreatic tumor cells reduced surface levels of MT1-MMP. Thus, in mesenchymal cells, loss of Dyn2 induces retention of MT1-MMP at the surface, leading to large scale matrix degradation through an MT1-MMP/MMP2-dependent mechanism. Excitingly, co-culture invasion experiments demonstrate that the matrix degradation induced in these stromal fibroblasts is sufficient to promote invasion by pancreatic tumor cells. These findings provide evidence for a novel matrix remodeling process conducted by mesenchymal cells that is substantially more effective than conventional invadopodia, distinct in structural organization, independent of the classical invadopodial machinery, and capable of promoting invasion by pancreatic tumor cells. Supported by NCI RO1 CA104125 to MAM. Citation Format: Hong Cao, Robbin Eppinga, Eugene Krueger, Jing Chen, Gina Razidlo, Mark A. McNiven. Differential mechanisms utilized by pancreatic tumor cells and associated fibroblasts to remodel stroma during metastatic invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3162. doi:10.1158/1538-7445.AM2014-3162