Ron knockdown and Ron monoclonal antibody IMC-RON8 sensitize pancreatic cancer to histone deacetylase inhibitors (HDACi).

Recepteur d\u27origine nantais (Ron) is overexpressed in a panel of pancreatic cancer cells and tissue samples from pancreatic cancer patients. Ron can be activated by its ligand macrophage stimulating protein (MSP), thereby activating oncogenic signaling pathways. Crosstalk between Ron and EGFR, c-Met, or IGF-1R may provide a mechanism underlying drug resistance. Thus, targeting Ron may represent a novel therapeutic strategy. IMC-RON8 is the first Ron monoclonal antibody (mAb) entering clinical trial for targeting Ron overexpression. Our studies show IMC-RON8 downmodulated Ron expression in pancreatic cancer cells and significantly blocked MSP-stimulated Ron activation, downstream Akt and ERK phosphorylation, and survivin mRNA expression. IMC-RON8 hindered MSP-induced cell migration and reduced cell transformation. Histone deacetylase inhibitors (HDACi) are reported to target expression of various genes through modification of nucleosome histones and non-histone proteins. Our work shows HDACi TSA and Panobinostat (PS) decreased Ron mRNA and protein expression in pancreatic cancer cells. PS also reduced downstream signaling of pAkt, survivin, and XIAP, as well as enhanced cell apoptosis. Interestingly, PS reduced colony formation in Ron knockdown cells to a greater extent than Ron scramble control cells in colony formation and soft agarose assays. IMC-RON8 could also sensitize pancreatic cancer cells to PS, as reflected by reduced colony numbers and size in combination treatment with IMC-RON8 and PS compared to single treatment alone. The co-treatment further reduced Ron expression and pAkt, and increased PARP cleavage compared to either treatment alone. This study suggests the potential for a novel combination approach which may ultimately be of value in treatment of pancreatic cancer

Identification of a Novel TGFβ/PKA Signaling Transduceome in Mediating Control of Cell Survival and Metastasis in Colon Cancer

Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFβ tumor suppressor activities in the metastatic process is essentially unknown.Utilizing in vitro and in vivo techniques, we have shown that loss of TGFβ tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFβ receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFβ/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFβ/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFβ receptor restoration with reactivation of the TGFβ/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival.This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFβ function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFβ signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFβ/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors

Panobinostat downregulated Ron mRNA, protein expression and decreased downstream signaling through pAkt, XIAP, and survivin.

<p>A) Pancreatic cancer cells were treated with different concentrations of PS for 8h, 24h, and 48 hours. Western blot for Ron was performed. β actin served as a loading control. B) Cells were treated with TSA with IB for Ron. Substantial number of cells were dead (about 80%) in 1000nM TSA-treated Capan-1 cells at 48 hours. C) Cells were treated with 50nM of PS for 8, 24 and 48 hours. RT- qPCR was performed on total RNA to determine mRNA level of Ron. GAPDH mRNA was used as an internal control. D) Cells were treated with different concentrations of PS at the indicated time points. Western blot for pAkt, XIAP and survivin expression. B-actin was used a loading control.</p

Panobinostat reduced cell growth and increased apoptosis through induction of cell cycle regulator p21 and caspase activity, respectively.

<p>Cells were treated with different concentrations of PS, A) cell growth was measured by MTT; B) cell apoptosis was measured by DNA fragmentation; C) Western blots for p21, and caspase activity (PARP and caspase 9 cleavages). B-actin was used as a loading control.</p

Mechanisms underlie IMC-RON8 sensitization of pancreatic cancer cells to Panobinostat.

<p>A) Ron expression after co-treatment of Capan-1, L3.6pl and CFPAC-1 cells with IMC-RON8 and PS. B) Effect of co-treatment of IMC-RON8 and PS on pAkt and PARP cleavage. β-actin was used as a loading control.</p

Ron knockdown or Ron mAb IMC-RON8 sensitized pancreatic cancer cells to Panobinostat.

<p>A) L3.6pl cells were stably transfected with Ron SC and shRNA plasmids. Ron KD clones A6 and B21 were selected for clonogenic assay: L3.6pl Ron SC and KD clones A6 and B21 were treated on day 2 with PS at indicated concentrations for 48 hours. After washing, cells were cultured for an additional 10 days in 10% FBS containing medium. Cell colonies were visualized after MTT staining and quantified in DMSO. B). Anchorage-independent growth of L3.6pl SC and Ron KD clones treated by PS was determined as described in Materials and Methods. Soft argrose assay was also performed for C) L3.6pl cells and D) CFPAC-1 cells treated with either IMC-RON8 or PS alone or their combinations.</p

IMC-RON8 downmodulated Ron expression and inhibited MSP-dependent Ron phosphorylation and downstream signaling in pancreatic cancer cells.

<p>A) Effect of IMC-RON8 (100nM) on Ron expression. Western blot for Ron. β-actin served as a loading control. B) Effect of IMC-RON8 on MSP-induced Ron activation. Cells were serum starved overnight and treated with IMC-RON8 for 1 hour followed by MSP stimulation for half hour. Cell lysates were subjected to IP, and IB for pRon. C) Effect of IMC-RON8 on downstream signaling. Same treatment as B). Western blot for pERK and pAkt. D) Ron activation and survivin mRNA expression. Capan-1 and CFPAC-1 cells were treated with IMC-RON8 followed by MSP stimulation for 12 hours after serum starvation for overnight. RT- qPCR was performed on total RNA to determine mRNA level of survivin. GAPDH mRNA was used as an internal control.</p