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

Abstract

BackgroundUnderstanding 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.Methodology/Principal FindingsUtilizing 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.Conclusion/SignificanceThis 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.

Protein Kinase A Modulates Transforming Growth Factor-β Signaling through a Direct Interaction with Smad4 Protein

“…Inhibition of PKA has also been previously found to attenuate TGF␤-induced stimulation of CREB phosphorylation and fibronectin gene expression (17,22). Recently, cross-talk between the TGF␤ and PKA signaling pathways has been found to be important in colon cancer cell survival and metastasis through regulation of survivin and XIAP signaling (23). We previously identified a novel mechanism of PKA activation by TGF␤ via the formation of a trimeric complex composed of activated Smad3, Smad4, and the regulatory subunit of PKA, with the resulting release of the catalytic subunit from the PKA holoenzyme (24).…”

Pharmacological targeting of AKAP-directed compartmentalized cAMP signalling

“…Such global uncoupling of PKA from AKAPs is not generally an option. Recently, contradictory roles of PKA/AKAP signalling were demonstrated in colon carcinoma cells; AKAP149 (D-AKAP1, AKAP1) was shown to be required for TGFβ-mediated PKA activation leading to apoptosis in response to cellular stress, such as oxidative stress, radiation, nutrient deprivation as well as cellular damage [237]. In contrast, a recently characterized AKAP, Praja2…”

Anti-tumor Activity of IGF-1R Kinase Inhibitor PQIP in Colon Cancer

“…The GEO human colon carcinoma cell line [29][30][31] was continuously maintained in a chemically defined serum-free medium. The standard maintenance medium (designated "SF") consisted of McCoy's 5A medium (Sigma, St. Louis, MO) supplemented with pyruvate, vitamins, amino acids, antibiotics, insulin (20 µg/ml, Sigma), transferrin (4 µg/ml, Sigma) and epidermal growth factor (EGF) (10 ng/ml, R&D Systems, Minneapolis, MN).…”

“…This experiment was carried as described previously (30) . Briefly, GEO cells were washed twice with cold PBS and harvested in TNESV lysis buffer (50 mMTris (pH 7.4), 100 mM NaCl, 1% NP-40, 2 mM EDTA, 0.1% SDS, 50 mM NaF, 10 mM Na 3 VO 4, 1 mM PMSF, 25 µg/ml β-glycerophosphate and one protease inhibitor cocktail tablet from Roche).…”

“…Portions of the tissues were placed in 10% formalin solution for 24 hours or were dissected and Citation: Chowdhury S, Dominguez I, Sharratt E, Spernyak J, Brattain MG, et al (2013) field at 20x magnification. The proliferation rates were determined by Ki-67 staining (Dako Corporation) semi-quantitatively as previously described [30]. …”