Accounting for an estimated ~70,000 new diagnoses and ~8,800 deaths in 2011, malignant melanoma is the most lethal skin cancer, representing ~8% of total cancers cases in the United States. Elucidation of the molecular mechanisms that drive its development, progression and therapeutic resistance is urgently needed. Activating Transcription Factor 2 (ATF2) is an AP1 transcription factor that functions divergently as an oncogene in melanoma and as a tumor suppressor in nonmalignant skin cancers. How ATF2 plays both functions has remained unclear. I recently found that Protein Kinase C, isoform epsilon (PKCepsilon) phosphorylates ATF2 on a novel phosphoacceptor site (T52), promoting its nuclear localization and transcriptional activation, conferring resistance to genotoxic stress. Thi phosphorylation blocks the ability of ATF2 to translocate to the mitochondria during genotoxic stress. At the mitochondrial outer membrane, ATF2 induces mitochondrial membrane leakage by perturbing hexokinase1 and voltage-dependent anion channel 1-(HK1:VDAC1) containing complexes, and activating pro-apoptotic Bcl2 protein, Bax. Specifically how PKCepsilon phosphorylation affects the transcriptional and non-transcriptional DNA damage response functions of ATF2 is not known. Precisely how ATF2 activates Bax and alters HK1:VDAC1 complexes is not clear. Furthermore, I have observed ATF2 in the cytosol during contexts other than genotoxic stress. Its precise function and biological ramifications (e.g., mitochondrial/metabolic changes) during such contexts is not known.
In Aim 1, I propose to determine how phosphorylation by PKCepsilon modulates ATF2 transcriptional activity by modulating its interaction with transcriptional regulators and AP1 partners, as well as its non-transcriptiona function in DNA damage response. I will also investigate how PKCepsilon affects ATF2 transcriptional output programming by investigating hits identified in gene expression microarray analyses that I have now performed on ATF2 mutants that mimic phosphorylation by PKCepsilon in the presence or absence of genotoxic stress. I have found that PKCepsilon phosphorylated ATF2 represses the expression of Interferon Beta 1 (IFNB1) and related downstream targets. IFNB1related signaling is known to suppress cellular proliferation, and its administration can sensitize cancer cells to chemotherapeutics. My preliminary data suggests that melanomas might develop resistance to genotoxic stress by suppressing IFNB1 expression through PKCepsilon ATF2mediated signaling. This raises the exciting notion that the therapeutic targeting of ATF2 in melanomas might derepress IFNB1 expression, rendering the cells sensitive to genotoxic stress (such as that exerted by frontline chemotherapeutic melanoma treatments). I will further investigate how PKCepsilon ATF2 suppresses IFNB1 expression during genotoxic stress. Detailed mechanistic studies of such pathways identified from my expression profiling studies will identify functional clusters of PKCepsilon ATF2regulated genes that are critical for melanoma development and/or therapeutic resistance. I will develop a syngeneic xenograft mouse melanoma model to assess how PKCepsilon phosphoregulation of ATF2 drives melanoma.
In Aim 2, I propose to determine how specifically ATF2 activates Bcl2 proteins and modulates HK1:VDAC1 complexes to promote mitochondrial membrane leakage. I will also identify and characterize non-genotoxic stress, physiological conditions, where we have observed ATF2 mitochondrial localization without cell death. I will characterize the resulting biological consequences, such as altered mitochondrial respiration or metabolism, and begin investigation of the molecular mechanism(s) underlying those changes that are mediated by ATF2. By determining how PKCepsilon alters ATF2 transcriptional activity and control of specific downstream genes that are critical for melanoma development and resistance, investigation from Aim 1 may be able to identify novel therapeutic modalities for melanoma. My proposed studies in Aim 2 will provide a better understanding of how ATF2 regulates of the integrity of the mitochondrial outer membrane, both during stress and physiological conditions. The K99/R00 award would allow for me to conduct my proposed studies under the continued guidance of my current mentor, Dr. Ze'ev Ronai, together with a panel of co-mentors who are renowned experts in melanoma and skin cancer biology, transcriptional regulation, PKCmediated signaling, and mitochondrial dynamics and cell death. During the mentored phase, I will complete several arms of Aims 1 and 2 at the Sanford-Burnham Medical Research Institute. During the R00 independent phase, I will independently continue my research at another academic or nonprofit research institution. My R00 phase investigation will further our understanding of how PKCepsilon regulated ATF2 transcription drives melanoma progression and resistance via regulation of IFNB1 (Aim 1 subaim iv and Aim 2, which will begin in the K99 phase and be completed within the R00 phase). I will then focus on other functional gene clusters identified from the expression array analyses in Aim 1. I will further investigate the metabolic ramifications of mitochondrial ATF2 identified in Aim 2. I believe that with my extensive biochemistry and cell biology background, being awarded a K99/R00 grant will promote and expedite the further development of my scientific and technical expertise, and my transition into a successful, independent research faculty in transcriptional control, protein signal transduction, melanoma and cancer.
The proposed studies will: (1) determine how PKCepsilon regulates the nuclear transcriptional and non-transcriptional functions of ATF2 to promote melanoma development and progression, and (2) to clarify the precise mechanism by which ATF2 regulates mitochondrial function. My proposed study represents an unprecedented opportunity to elucidate novel PKCepsilon-ATF2regulated mechanisms driving melanoma progression and development, as well as novel regulation of mitochondrial integrity, and will likely identify new signaling modules that are critical for melanoma biology and resistance. As PKCepsilon expression was recently found to confer resistance to anti-melanoma therapy (mutant BRAF inhibitor), targeting of PKCepsilon ATF2regulated pathways will have implications for future targeted therapies.