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Constantine A. Stratakis - One of the best experts on this subject based on the ideXlab platform.
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Cyclic AMP‐dependent protein kinase catalytic subunit A (PRKACA): the expected, the unexpected, and what might be next
The Journal of Pathology, 2018Co-Authors: Constantine A. StratakisAbstract:Protein kinase A (PKA) or cyclic-AMP (cAMP)-dependent kinase was among the first serine-threonine kinases to be molecularly and functionally characterized. For years, it was investigated as the enzyme that mediates cAMP functions in almost all cell systems and organisms studied to date. Despite PKA's critical role in signaling and the long history of investigations of cAMP in oncogenesis (dating back to the 1970s), it was not until relatively recently that PKA defects were found to be directly involved in tumor predisposition. First, PKA's main regulatory subunit, PRKAR1A, was found to be mutated in Carney complex, a genetic syndrome that predisposes to heart tumors (cardiac myxomas) and a variety of other lesions of the endocrine system, including the adrenal cortex, and several cancers, including liver carcinoma. Then, PKA's main catalytic subunit, PRKACA, was found to be mutated in sporadic adrenal tumors and fibrolamellar liver carcinoma. Not surprisingly, therefore, a new research study published in The Journal of Pathology showed PRKACA mutations in sporadic cardiac myxomas. The real question is what other pathologies will be found to be due to PRKACA (or other PKA subunit) defects. The possibilities abound and may show the way for a totally new class of medications that target cAMP signaling to be useful in fighting the corresponding tumors. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.
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lipofuscin accumulation in cortisol producing adenomas with and without PRKACA mutations
Hormone and Metabolic Research, 2017Co-Authors: Anna Angelousi, Eva Szarek, Vincent Shram, Electron Kebebew, Martha Quezado, Constantine A. StratakisAbstract:The adrenal cortex accumulates lipofuscin granules with age. Lipofuscin accumulation is also seen in adrenocortical tumors associated with Cushing syndrome (CS), particularly those with PRKAR1A mutations, such as in primary pigmented nodular adrenocortical disease (PPNAD). We investigated the presence of lipofuscin in cortisol-producing adenomas (CPAs) responsible for CS with and without the PRKACA (pLeu206Arg) somatic mutation. Ten paraffin-embedded sections of CPAs from cases with overt CS with (n=4) and without (n=6) a PRKACA mutation were microscopically examined through three detection methods, the hematoxylin-Eosin (H & E) staining, the Fontana Masson (FM) staining using light microscopy, and lipofuscin autofluorescence, using confocal laser scanning microscopy (CLSM). Sections were examined quantitatively according to the intensity of the pigmentation, as well as qualitatively based on the total number of granular pigments at all visual fields per tissue slide. Tissues from CPAs were compared to peritumoral adjacent tissues (n=5), to Conn adenomas (n=4), and PPNAD (n=3). CPAs had significantly higher number of lipofuscin-pigment granules compared to peritumoral adrenal tissue and Conn adenomas (46.9±9.5 vs. 3.8±4.8, p=0.0001). The presence of the PRKACA mutation did not increase the chances of pigmentation in the form of lipofuscin granules within CPAs associated with CS. Thus, all CPAs leading to CS accumulate lipofuscin, which presents like pigmentation sometimes seen macroscopically but always detected microscopically. PPNAD caused by PRKAR1A mutations is the best known adrenal lesion leading to CS associated with intense lipofuscin pigmentation and this was confirmed here; CPAs harboring PRKACA mutations did not have statistically significantly more pigmentation than CPAs without mutation, but a larger study might have shown a difference.
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Studies of mice with cyclic AMP-dependent protein kinase (PKA) defects reveal the critical role of PKA's catalytic subunits in anxiety.
Behavioural Brain Research, 2016Co-Authors: George Briassoulis, Margaret F. Keil, Bilal Naved, Matthew F. Starost, Maria Nesterova, Nirmal Gokarn, Anna Batistatos, T. John Wu, Constantine A. StratakisAbstract:Abstract Cyclic adenosine mono-phosphate-dependent protein kinase (PKA) is critically involved in the regulation of behavioral responses. Previous studies showed that PKA’s main regulatory subunit, R1α, is involved in anxiety-like behaviors. The purpose of this study was to determine how the catalytic subunit, Cα, might affect R1α’s function and determine its effects on anxiety-related behaviors. The marble bury (MB) and elevated plus maze (EPM) tests were used to assess anxiety-like behavior and the hotplate test to assess nociception in wild type (WT) mouse, a Prkar1a heterozygote (Prkar1a +/− ) mouse with haploinsufficiency for the regulatory subunit (R1α ), a PRKACA heterozygote (PRKACA +/− ) mouse with haploinsufficiency for the catalytic subunit (Cα), and a double heterozygote mouse ( Prkar1a +/− / PRKACA +/− ) with haploinsufficiency for both R1α and Cα. We then examined specific brain nuclei involved in anxiety. Results of MB test showed a genotype effect, with increased anxiety-like behavior in Prkar1a +/− and Prkar1a +/− / PRKACA +/− compared to WT mice. In the EPM, Prkar1a +/− spent significantly less time in the open arms, while PRKACA +/− and Prkar1a +/− /PRKACA +/− mice displayed less exploratory behavior compared to WT mice. The loss of one Prkar1a allele was associated with a significant increase in PKA activity in the basolateral (BLA) and central (CeA) amygdala and ventromedial hypothalamus (VMH) in both Prkar1a +/− and Prkar1a +/− /PRKACA +/− mice. Alterations of PKA activity induced by haploinsufficiency of its main regulatory or most important catalytic subunits result in anxiety-like behaviors. The BLA, CeA, and VMH are implicated in mediating these PKA effects in brain.
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Carney Complex: an update
European Journal of Endocrinology, 2015Co-Authors: Ricardo G. Correa, Paraskevi Salpea, Constantine A. StratakisAbstract:Carney complex (CNC) is a rare autosomal dominant syndrome, characterized by pigmented lesions of the skin and mucosa, cardiac, cutaneous and other myxomas and multiple endocrine tumors. The disease is caused by inactivating mutations or large deletions of the PRKAR1A gene located at 17q22–24 coding for the regulatory subunit type I alpha of protein kinase A (PKA) gene. Most recently, components of the complex have been associated with defects of other PKA subunits, such as the catalytic subunits PRKACA (adrenal hyperplasia) and PRKACB (pigmented spots, myxomas, pituitary adenomas). In this report, we review CNC, its clinical features, diagnosis, treatment and molecular etiology, including PRKAR1A mutations and the newest on PRKACA and PRKACB defects especially as they pertain to adrenal tumors and Cushing’s syndrome.
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PRKACA the catalytic subunit of protein kinase a and adrenocortical tumors
Frontiers in Cell and Developmental Biology, 2015Co-Authors: Annabel Berthon, Eva Szarek, Constantine A. StratakisAbstract:Cyclic-AMP (cAMP)-dependent protein kinase (PKA) is the main effector of cAMP signaling in all tissues. Inactivating mutations of the PRKAR1A gene, coding for the type 1A regulatory subunit of PKA, are responsible for Carney complex and primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A inactivation and PKA dysregulation have been implicated in various types of adrenocortical pathologies associated with ACTH-independent Cushing syndrome (AICS) from PPNAD to adrenocortical adenomas and cancer, and other forms of bilateral adrenocortical hyperplasias (BAH). More recently, mutations of PRKACA, the gene coding for the catalytic subunit C alpha (Cα), were also identified in the pathogenesis of adrenocortical tumors. PRKACA copy number gain was found in the germline of several patients with cortisol-producing BAH, whereas the somatic Leu206Arg (c.617A>C) recurrent PRKACA mutation was found in as many as half of all adrenocortical adenomas associated with AICS. In vitro analysis demonstrated that this mutation led to constitutive Cα activity, unregulated by its main partners, the PKA regulatory subunits. In this review, we summarize the current understanding of the involvement of PRKACA in adrenocortical tumorigenesis, and our understanding of PKA’s role in adrenocortical lesions. We also discuss potential therapeutic advances that can be made through targeting of PRKACA and the PKA pathway.
Z C Wang - One of the best experts on this subject based on the ideXlab platform.
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PRKACA mediates resistance to HER2-targeted therapy in breast cancer cells and restores anti-apoptotic signaling
Oncogene, 2015Co-Authors: S E Moody, A C Schinzel, S Singh, F Izzo, M R Strickland, S R Thomas, J S Boehm, Z C Wang, W C HahnAbstract:Targeting HER2 with antibodies or small molecule inhibitors in HER2-positive breast cancer leads to improved survival, but resistance is a common clinical problem. To uncover novel mechanisms of resistance to anti-HER2 therapy in breast cancer, we performed a kinase open reading frame screen to identify genes that rescue HER2 -amplified breast cancer cells from HER2 inhibition or suppression. In addition to multiple members of the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) signaling pathways, we discovered that expression of the survival kinases PRKACA and PIM1 rescued cells from anti-HER2 therapy. Furthermore, we observed elevated PRKACA expression in trastuzumab-resistant breast cancer samples, indicating that this pathway is activated in breast cancers that are clinically resistant to trastuzumab-containing therapy. We found that neither PRKACA nor PIM1 restored MAPK or PI3K activation after lapatinib or trastuzumab treatment, but rather inactivated the pro-apoptotic protein BAD, the BCl-2-associated death promoter, thereby permitting survival signaling through BCL-XL. Pharmacological blockade of BCL-XL/BCL-2 partially abrogated the rescue effects conferred by PRKACA and PIM1, and sensitized cells to lapatinib treatment. These observations suggest that combined targeting of HER2 and the BCL-XL/BCL-2 anti-apoptotic pathway may increase responses to anti-HER2 therapy in breast cancer and decrease the emergence of resistant disease.
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PRKACA mediates resistance to her2 targeted therapy in breast cancer cells and restores anti apoptotic signaling
Oncogene, 2015Co-Authors: A C Schinzel, S Singh, F Izzo, M R Strickland, S R Thomas, J S Boehm, Z C Wang, Susan MoodyAbstract:PRKACA mediates resistance to HER2-targeted therapy in breast cancer cells and restores anti-apoptotic signaling
Ute I Scholl - One of the best experts on this subject based on the ideXlab platform.
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PRKACA mutations in cortisol producing adenomas and adrenal hyperplasia a single center study of 60 cases
European Journal of Endocrinology, 2015Co-Authors: Anne Thiel, Annacarinna Reis, M Haase, M Schott, H S Willenberg, Ute I SchollAbstract:Objective: Cortisol excess due to adrenal adenomas or hyperplasia causes Cushing’s syndrome. Recent genetic studies have identified a somatic PRKACA L206R mutation as a cause of cortisol-producing adenomas. We aimed to compare the clinical features of PRKACA-mutant lesions with those of CTNNB1 mutations, and to search for similar mutations in unilateral hyperplasia or tumors co-secreting aldosterone. Design, patients, and methods: In this study, 60 patients with cortisol excess who had adrenalectomies at our institution between 1992 and 2013 were assessed, and somatic mutations were determined by Sanger sequencing. A total of 36 patients had overt Cushing’s syndrome, the remainder were subclinical: 59 cases were adenomas (three bilateral) and one was classified as hyperplasia. Four tumors had proven co-secretion of aldosterone. Results: Among cortisol-secreting unilateral lesions without evidence of co-secretion (nZ52), we identified somatic mutations in PRKACA (L206R) in 23.1%, CTNNB1 (S45P, S45F) in 23.1%, GNAS (R201C) in 5.8%, and CTNNB1CGNAS (S45P, R201H) in 1.9%. PRKACA and GNAS mutations were mutually exclusive. Of the co-secreting tumors, two (50%) had mutations in KCNJ5 (G151R and L168R). The hyperplastic gland showed a PRKACA L206R mutation, while patients with bilateral adenomas did not have known somatic mutations. PRKACA-mutant lesions were associated with younger age, overt Cushing’s syndrome, and higher cortisol levels vs non-PRKACA-mutant or CTNNB1-mutant lesions. CTNNB1 mutations were more significantly associated with right than left lesions. Conclusions: PRKACA L206R is present not only in adenomas, but also in unilateral hyperplasia and is associated with more severe autonomous cortisol secretion. Bilateral adenomas may be caused by yet-unknown germline mutations.
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recurrent activating mutation in PRKACA in cortisol producing adrenal tumors
Nature Genetics, 2014Co-Authors: Ute I Scholl, James M Healy, Murim Choi, Manju L Prasad, Carol Nelsonwilliams, Reju Korah, Anna Carinna Suttorp, Dimo Dietrich, John W Kunstman, M HaaseAbstract:Richard Lifton and colleagues identify a recurrent activating mutation in PRKACA, which encodes the catalytic subunit of protein kinase A, in cortisol-producing adrenal tumors. They further show that the mutation results in loss of binding by the regulatory subunit PRKAR1A, leading to increased phosphorylation of downstream targets.
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corrigendum recurrent activating mutation in PRKACA in cortisol producing adrenal tumors
Nature Genetics, 2014Co-Authors: Ute I Scholl, James M Healy, Murim Choi, Manju L Prasad, Carol Nelsonwilliams, John W Kuntsman, Reju Korah, Anna Carinna Suttorp, Dimo Dietrich, M HaaseAbstract:Corrigendum: Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors
Khashayar Vakili - One of the best experts on this subject based on the ideXlab platform.
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abstract b37 assessing dna damage response pathway proteins atm and p53 in fibrolamellar hepatocellular carcinoma with PRKACA dnajb1 fusion protein
Cancer Research, 2018Co-Authors: Anju Karki, Khashayar Vakili, Antonio R PerezataydeAbstract:Introduction: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare variant of liver cancer that is prevalent in younger patients between 10-35 years of age and is usually not associated with underlying cirrhosis. Overall outcomes tend to be poor due to lack of effective chemotherapy. Recently, a gene fusion transcript has been identified in a significant proportion of FL-HCC whereby PRKACA, a cAMP-associated protein, and heat shock protein DNAJB1 fuse following a deletion of 400kb region. In our previous study, immunoblot analysis of p53, a tumor-suppressor protein, was significantly upregulated in FL-HCC samples in comparison to normal liver tissues. p53 is a downstream target of ATM, which is a key regulator of DNA damage response (DDR) pathway. Therefore, we hypothesized that increased p53 protein expression may be associated with ATM activation in FL-HCC. Methods: We analyzed 3 tumor and non-neoplastic FL-HCC patient samples containing DNAJB1-PRKACA fusion protein using immunohistochemistry (IHC) on paraffin-embedded sections with anti-ATM, anti-phosphorylated ATM (S1981), anti-p53, and anti-phospho-p53 (S15) antibodies. Immunoblot analysis was performed for cleaved PARP protein to assess the apoptotic cells in FL-HCC samples. ImageJ software was utilized to quantify the nuclear, cytoplasmic/mitochondrial localization of each protein. Results: Nuclear ATM expression was significantly increased in tumor samples compared to normal liver tissues in 3/3 FL-HCC patients (p=1.43E-04, p=1.51E-07 and 1.68E-05). Nuclear expression of activated (phosphorylated) ATM (p-ATM) was significantly upregulated in 2/3 tumor compared to the normal samples (p=9.47E-09 and p=0.00). Interestingly, the tumor sample lacking nuclear phospho-ATM was the only tumor sample with a significant expression of p-ATM in the mitochondrial/cytoplasmic compartment. In assessing the expression pattern of p-53, 2/3 patients revealed statistically significant increase in total p-53 compared to normal liver sample. While nuclear phospho-p53 expression was not significantly altered in any of the three tumor samples compared to non-neoplastic liver tissue, there was a statistically significant increase in the activated form of p53 (phospho-p53) expression in the cytoplasmic region in 2/3 FLHCC samples compared to normal. Coincidentally, the same 2 patients who demonstrated increased total nuclear ATM and phospho-ATM showed increased cytoplasmic p53 and phosphorylated p53. Immunoblot analysis for cleaved-PARP revealed no PARP activation in FL-HCC tumor samples. Conclusion: The expression pattern of total ATM and phospho-ATM in FL-HCC patients with PRKACA-DNAJB1 suggests that the DDR pathway may be activated in these tumors. However, increases in p53 protein expression and phosphorylated (S15) p53 are not associated with apoptosis given lack of increase in PARP cleavage. Further studies aimed at understanding the mechanisms preventing p53-induced apoptosis or DNA repair are therefore necessary. Citation Format: Anju Karki, Khashayar Vakili, Antonio R. Perez-Atayde. Assessing DNA damage response pathway proteins ATM and p53 in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B37.
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abstract 4150 mdm4 expression is increased in fibrolamellar hepatocellular carcinoma with PRKACA dnajb1 fusion protein
Cancer Research, 2018Co-Authors: Anju Karki, Michael P Laquaglia, Juan Putra, Antonio R Perezatayde, Khashayar VakiliAbstract:Introduction: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare variant of hepatocellular carcinoma (HCC) that preferentially affects young adults with no underlying liver disease. Overall patient outcome is poor due to the lack of effective treatment options. Recent studies have shown a 400 kb pair deletion resulting in DNJAB1-PRKACA fusion transcript and protein in majority of FL-HCC tumors. The exact oncogenic mechanism of this fusion protein is yet to be elucidated. In our previous studies we had noted upregulation of p53 and phosphorylated p53 in tumor samples compared to normal liver tissue without expected p53-induced apoptosis. Since MDM4 (a.k.a. MDMX or HDMX), a known negative regulator of p53, is overexpressed in various human cancers, we sought to examine its role in dysregulation of p53 signaling pathway in FL-HCC containing PRKACA-DNAJB1 fusion. Methods: We analyzed 7 FL-HCC tumors containing DNAJB1-PRKACA fusion protein and 5 non-neoplastic samples. Immunohistochemistry (IHC) on formalin-fixed and paraffin-embedded sections was performed using primary anti-MDM4 antibody. Diaminobenzidine (DAB) substrate kit was utilized to detect the signal and the slides were counterstained with hematoxylin. The nuclear MDM4 expression was assessed using immune reactive score (IRS) in a blinded manner. The IRS gives a range of 0-12 as a product of multiplication between positive cells proportion score (0-4) and staining intensity score (0-3). We also analyzed transcript levels of MDM4 from tumor and non-neoplastic liver samples on 4/7 patients. Reactions were performed in duplicates and MDM4 was normalized to GAPDH. Results: We found that the percentage of positive MDM4 cells ranged from 0 to 80 in normal liver samples (5/7 patients) and 10 to 100 in tumor samples (7/7 patients). The IRS score of MDM4 ranged from 0 to 6 in non-neoplastic liver tissue and 2 to 12 in tumor tissue. Statistically, the IRS score of MDM4 protein expression was significantly upregulated in tumor samples in comparison to non-neoplastic samples (P=0.0098). Additionally, the MDM4 transcript levels in 2/4 tumors examined were increased by 2- and 180-fold, respectively, compared to non-neoplastic liver tissue. Conclusion: The overexpression of MDM4 protein in FL-HCC patients with PRKACA-DNAJB1 suggests that the p53 effector function of DNA repair or induction of apoptosis may be dysregulated by increased MDM4 expression. Further studies targeted at blocking MDM4 inhibition of p53 activity in FL-HCC will be important in determining its contribution to tumor cell survival and proliferation. Citation Format: Anju Karki, Juan Putra, Michael LaQuaglia, Antonio Perez-Atayde, Khashayar Vakili. MDM4 expression is increased in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4150.
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abstract 3514 assessing the role of creb in fibrolamellar hepatocellular carcinoma with PRKACA dnajb1 fusion protein
Cancer Research, 2017Co-Authors: Anju Karki, Michael P Laquaglia, Amanda M Dios, Ghazaleh Sadrivakili, Khashayar VakiliAbstract:Introduction: Fibrolamellar Hepatocellular Carcinoma (FLHCC) is a rare form of liver cancer that occurs predominantly in adolescents or young adults. Curing or prolonging the lives of patients with FLHCC is extremely challenging. Despite aggressive surgical resections of primary tumors as well as metastatic lesions, outcomes tend to be poor due to lack of effective chemotherapy. Previous studies have demonstrated the presence of PRKACA-DNAJB1 fusion protein in the majority of FLHCC patients. DNAJB1 is a heat shock protein and PKA is a kinase that phosphorylates numerous substrates including cAMP-regulatory element-binding protein (CREB), a proto-oncogenic transcription factor. Therefore, we sought to examine whether the presence of the fusion protein in FLHCC is associated with increased CREB phosphorylation. Methods: We analyzed tumor and matched non-neoplastic liver tissue from 7 pediatric patients with a pathologic diagnosis of FLHCC. We assessed the presence of the PRKACA-DNAJB1 fusion protein using PKA antibody, as well as total CREB and phosphorylated (p-CREB) using immunoblot analyses with specific antibodies. Results: Immunoblot analysis using anti-PKA antibody confirmed the occurrence of PRKACA-DNAJB1 fusion transcripts in 6/7 (86%) FLHCC tumor samples. Immunoblot analyses of the 6 patients with the fusion protein revealed a trend towards a decrease in p-CREB expression in tumor samples compared to non-neoplastic tissue controls (p=0.15). Conclusions: The pattern of p-CREB in FLHCC with PRKACA-DNAJB1 fusion protein suggests that the fusion protein does not exert its proto-oncogenic effects through the transcriptional activity of CREB. Citation Format: Anju Karki, Michael J. LaQuaglia, Amanda M. Dios, Ghazaleh Sadri-Vakili, Khashayar Vakili. Assessing the role of CREB in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein [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 3514. doi:10.1158/1538-7445.AM2017-3514
Susan Moody - One of the best experts on this subject based on the ideXlab platform.
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PRKACA mediates resistance to her2 targeted therapy in breast cancer cells and restores anti apoptotic signaling
Oncogene, 2015Co-Authors: A C Schinzel, S Singh, F Izzo, M R Strickland, S R Thomas, J S Boehm, Z C Wang, Susan MoodyAbstract:PRKACA mediates resistance to HER2-targeted therapy in breast cancer cells and restores anti-apoptotic signaling
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Overcoming Resistance to Trastuzumab in HER2-Amplified Breast Cancers
2011Co-Authors: Susan MoodyAbstract:Abstract : The receptor tyrosin kinase HER2 is amplified and/or overexpressed in 25-30% of all breast cancers. Blockade of HER2 with drugs such as trastuzumab or lapatinib has led to clinical benefit in patients with both metastatic and early-stage HER2-amplified breast cancer. However, resistance and disease progression always occurs in patients with metastatic disease, and many patients with early-stage breast cancer experience recurrences despite adjuvant treatment with one of these agents. To identify novel mechanisms of resistance to anti-HER2 therapy, I conducted a screen for molecules whose forced expression in HER2-amplified breast cancer cells confers resistance to HER2 inhibition. I screened an open reading frame library of approximately 600 kinases and kinase-related molecules. I found that both activated HRAS and the catalytic subunit of Protein Kinase A (PRKACA) conferred significant resistance to both compound-mediated and genetic inhibition of HER2. Upon further mechanistic investigation, I found that activated HRAS fully restored phospo-ERK levels in the presence of either of two HER2 tyrosine kinase inhibitors, but did not restore phospho-AKT1 levels. By contrast, PRKACA expression provided resistance to anti-HER2 treatment, but did not restore phospho-ERK or phospho-AKT1 levels. The mechanisms by which resistance is conferred by PRKACA are under continued investigation.