Prostate cancer (PCa) is associated with chronic prostate inflammation resulting in activation of stress and pro-survival pathways that contribute to disease progression and chemoresistance. The stress oncoprotein lens epithelium-derived growth factor p75 (LEDGF/p75), also known as DFS70 autoantigen, promotes cellular survival against environmental stressors, including oxidative stress, radiation, and cytotoxic drugs. Furthermore, LEDGF/p75 overexpression in PCa and other cancers has been associated with features of tumor aggressiveness, including resistance to cell death and chemotherapy. We report here that the endogenous levels of LEDGF/p75 are upregulated in metastatic castration resistant prostate cancer (mCRPC) cells selected for resistance to the taxane drug docetaxel (DTX). These cells also showed resistance to the taxanes cabazitaxel (CBZ) and paclitaxel (PTX), but not to the classical inducer of apoptosis TRAIL. Silencing LEDGF/p75 effectively sensitized taxane-resistant PC3 and DU145 cells to DTX and CBZ, as evidenced by a significant decrease in their clonogenic potential. While TRAIL induced apoptotic blebbing, caspase-3 processing, and apoptotic LEDGF/p75 cleavage, which leads to its inactivation, in both taxane-resistant and -sensitive PC3 and DU145 cells, treatment with DTX and CBZ failed to robustly induce these signature apoptotic events. These observations suggested that taxanes induce both caspase-dependent and -independent cell death in mCRPC cells, and that maintaining the structural integrity of LEDGF/p75 is critical for its role in promoting taxane-resistance. Our results further establish LEDGF/p75 as a stress oncoprotein that plays an important role in taxane-resistance in mCRPC cells, possibly by antagonizing drug-induced caspase-independent cell death.

African American women suffer higher incidence and mortality of triple negative breast cancer (TNBC) than Caucasian women. TNBC is very aggressive, causing the worst clinical outcome. We previously demonstrated that tumors from these patients express high IGF-II and exhibit high activation of the IGF signaling pathways. IGF-II gene expression is imprinted (monoallelic), promotes tumor progression, and metastasis and regulates Survivin, a TNBC prognostic marker. Since BC mortality has increased among young Vietnamese women, we analyzed 48 (paired) TNBC samples from Vietnamese patients to assess IGF-II expression. We analyzed all samples by qrtPCR for identification of IGF-II heterozygosity and to determine allelic expression of the IGF-II gene. We also analyzed the tissues for proIGF-II and Survivin by RT-PCR and Western blotting. A total of 28 samples displayed IGF-II heterozygosity of which 78% were biallelic. Tumors with biallelic IGF-II gene expression exhibited the highest levels of proIGF-II and Survivin. Although 100% of these tissues corresponding normal samples were biallelic, they expressed significantly lower levels of or no proIGF-II and Survivin. Thus, IGF-II biallelic gene expression is differentially regulated in normal versus tumor tissues. We propose that intratumoral proIGF-II is dependent on the IGF-II gene imprinting status and it will promote a more aggressive TNBC.

IGF2 is essential in breast differentiation, lactation, tumor growth, and in breast cancer (BC) development and progression. This growth factor also inhibits apoptosis and promotes metastasis and chemoresistance, contributing to more aggressive tumors. We previously demonstrated that IGF2 protein levels are higher in BC tissues from African American women than in Caucasian women. We also showed that high IGF2 protein levels are expressed in normal breast tissues of African American women while little or no IGF2 was detected in tissues from Caucasian women. Others showed that decreased DNA methylation of the IGF2 gene leads to different BC clinical features. Thus, we designed this study to determine if differentially methylated regions of the IGF2 gene correspond to IGF2 protein expression in paired (Normal/Tumor) breast tissues and in BC cell lines. Methylation analysis was performed using Sodium Bisulphite Analysis and Methylation Sensitive Restriction Enzyme digestion methods. Our results show that a unique site in the INS-IGF2 region is hypermethylated in normal breast and hypomethylated in breast cancer. We designated this region the DVDMR. Furthermore, the methylation levels in the DVDMR significantly correlated with IGF2 protein levels. This novel DMR consists of 257bp localized in the INS-IGF2 gene. We propose that methylation of DVDMR represents a novel epigenetic biomarker that determines the levels of IGF2 protein expression in breast cancer. Since IGF2 promotes metastasis and chemoresistance, we propose that IGF2 levels contribute to BC aggressiveness. Validation of IGF2 as a biomarker will improve diagnosis and treatment of BC patients.

Triple-negative breast cancer (TNBC) is very aggressive, resistant to chemotherapy and more likely to relapse, causing the worst prognosis. African American (AA) women suffer higher incidence and mortality of TNBC due to the expression of high levels of Insulin Growth Factor 2 (IGF2) which promotes tumor progression, metastasis, and chemoresistance. Also, it has been established that functional mitochondria and mitochondrial DNA (mtDNA) are essential for cancer cell growth. Mutations and/or reductions in mtDNA copy number that alter the Oxidative Phosphorylation (OXPHOS) physiology are common features of TNBC. We have demonstrated that mtDNA content is lower in CRL-2335 AA TNBC cell line when compared to the CRL-2335 IGF2 knockout cell line. Thus, we propose that IGF2 regulates the mtDNA content. This study was designed to demonstrate if IGF2 regulates mitochondrial genes to determine the cell energy phenotype. An XFp analyzer was used to study the mitochondrial function in terms of OCR (Oxygen Consumption Rate/Mitochondrial Respiration) and ECAR (Extracellular Acidification Rate/ Glycolysis) in the wild type and IGF2 stable knockout of CRL-2335 AA TNBC cells. Real Time PCR was performed to study the gene expression pattern of IGF2, PGC1α and PGC1β. PGC1α and PGC1β are critical genes in the regulation of the mitochondrial biogenesis, thus, they are important in the cellular metabolic phenotype. Utilizing the Seahorse metabolic system we assessed cell energy phenotype and alterations in terms of mitochondrial respiration rate (OCR) % and Glycolysis (ECAR) %. Our preliminary results showed that the overall OCR and ECAR of the stressed CRL-2335 AA TNBC cells was altered according to the levels of IGF-II expressed. Furthermore, the results demonstrated that there was a metabolic shift in the CRL-2335 AA TNBC cells towards the glycolytic pathway when IGF2 was knockout in comparison to the wild type CRL-2335 cells. Also, IGF2 knockout cells showed higher gene expression rate of PGC1β as compared to the wild type. The above data confirms that IGF2 plays a critical role in determining the cell energy phenotype. Citation Format: Maria L. Pagan, Vinodh Kumar Radhakrishnan, Daisy De Leon. IGF2 regulates mitochondrial cell energy phenotype and biogenesis in TNBC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4421. doi:10.1158/1538-7445.AM2017-4421

Triple negative breast cancer (TNBC) is more likely to be advanced, poorly differentiated, larger, and present in women of younger age and of lower socioeconomic status. Moreover, TNBC patients have worse outcomes when treated with currently available adjuvant chemotherapy. In the United States, African American (AA) women have a lower incidence of breast cancer compared with Caucasian women (CA), but higher overall mortality. Our lab have demonstrated that BC tumors from AA patients express significantly higher levels of IGF-II and show a higher activation of the IGF signaling pathways (Kalla et.al., 2010a, 2010b). IGF-II levels regulate survival proteins and activate the ER signaling pathway promoting an aggressive tumor development. Thus we designed a study to determine if IGF-II regulates the progression of TNBC tumors. TNBC incidence is increasing in younger Vietnamese women. Thus, we designed a study to analyze the IGF-II expression in paired normal-tumor BC tissues from Vietnamese TNBC patients. Furthermore, we decided to characterize the IGF-II genomic imprinting status, to correlate IGF-II levels with Monoallelic or Biallelic IGF-II gene expression. A total of 24 paired BC samples obtained from ILSbio were analyzed by Western Blot, RT-PCR and gDNA PCR with Apa I restriction digestion. Our results showed that the IGF-II genomic imprinting status in all 24 pairs was the same in normal and tumor tissues from the same patient. The levels of IGF-II correlated with the imprinting status, i.e., Biallelic (BA; n=14)≫, Monoallelic with SNP (MAS; n=5)>, and Monoallelic non-ApaI SNP (MA; n=5)>. Higher IGF-II levels were expressed in tumor as compared to normal tissue from the same patient. The free proIGF-II protein levels were 6.5-15.22 fold higher in biallelic tumor tissues as compared to the proIGF-II levels detected in monoallelic tumor samples. The IGF-II mRNA levels were 0.4-1 fold higher in biallelic tumor tissues as compared to the proIGF-II levels detected in monoallelic tumor samples. We also assessed growth factor receptors; Insulin receptor A (IRA), Insulin receptor B (IRB), Vascular endothelial growth factor receptor (VEGFR), Estrogen receptor-1 (ESR1), Progesterone receptor (PGR) and Human epidermal growth factor receptor (ERBB2) status to determine if IGF-II monoallelic or biallelic dosage regulated the receptor levels. We conclude that biallelic IGF-II expression in TNBC tumors results in higher IGF-II mRNA and protein levels than in monoallelic samples. Furthermore, higher IGF-II resulted in increased expression and signaling activation of IRA, IRB, VEGFR, ESR1, PGR and ERBB2 growth factor receptors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3131. doi:1538-7445.AM2012-3131