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The p19ARF tumor suppressor limits ribosome biogenesis and responds to hyperproliferative signals to activate the p53 checkpoint response. Although its activation of p53 has been well characterized, the role of ARF in restraining nucleolar ribosome production is poorly understood. Here we report the use of a mass spectroscopic analysis to identify protein changes within the nucleoli of Arf-deficient mouse cells. Through this approach, we discovered that ARF limited the nucleolar localization of the RNA helicase DDX5, which promotes the synthesis and maturation of rRNA, ultimately increasing ribosome output and proliferation. ARF inhibited the interaction between DDX5 and nucleophosmin (NPM), preventing association of DDX5 with the rDNA promoter and nuclear pre-ribosomes. In addition, Arf-deficient cells transformed by oncogenic RasV12 were addicted to DDX5, because reduction of DDX5 was sufficient to impair RasV12-driven colony formation in soft agar and tumor growth in mice. Taken together, our findings indicate that DDX5 is a key p53-independent target of the ARF tumor suppressor and is a novel non-oncogene participant in ribosome biogenesis. Cancer Res; 71(21); 6708–17. ©2011 AACR.

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Multiplex immunoassays are valuable research tools for studying circulating cytokines. Here we evaluate the ability of the MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A (Cat. No. HCYTA-60K) 48-plex immunoassay to reproducibly measure cytokines in matched human serum and plasma samples from healthy donors. For comparison, three other bead-based cytokine multiplex kits, featuring many of the same analytes, were analyzed in parallel. Two different lots from each kit were tested with the same matched sample cohort and data were analyzed with Belysa® software. The HCYTA-60K kit was unique in its use of a fixed standard curve, resulting in superior consistency in curve performance between lots. Sample value ratios and correlation demonstrated remarkable consistency of serum and plasma measurement between lots of the HCYTA-60K kit. Further, detectability of additional sample types (urine, tears, milk, CSF, and saliva) was determined with the HCYTA-60K kit. Samples from healthy donors for each biofluid were tested to determine detectability. All analytes could be measured in at least one of the additional sample types. To characterize neuroinflammation during progressive stages of neurological disease, CSF cytokines were measured in samples from mild cognitive impairment, Alzheimer’s Disease, and healthy controls. Our study illustrates the reliability of the MILLIPLEX® HCYTA-60K kit for reproducible measurement of cytokines in serum and plasma between different kit lots. Additionally, we characterize its ability to measure cytokines in urine, tears, milk, CSF, and saliva. These results highlight the utility and robustness of the HCYTA-60K multiplex panel for the quantification of cytokines in diverse biofluids.

Ras proteins (HRAS, KRAS, and NRAS) are small GTPases that function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states. Ras-GTP activates downstream pathways, including MAPK, by binding to Raf kinase and phosphatidylinositol 3-kinase (PI3K) to promote cellular proliferation, survival, growth and differentiation. Oncogenic mutations in Ras are found in approximately 25% of human cancers. Furthermore, 90% of pancreatic cancers harbor KRAS mutations with 80% of KRAS mutations occurring at codon 12 with G12V and G12D being the most prevalent. The Ras-Raf-MEK pathway can also be activated downstream of Ras. BRAF is a well-established oncogene, with the BRAF V600E mutation being prevalent in melanoma. While targeting this BRAF oncoprotein has had therapeutic efficacy, treating cancers driven by Ras oncoproteins remains an urgent unmet clinical need. We developed a novel multiplex immunoassay capable of simultaneously detecting total Ras, RasG12V, RasG12D, phospho-MEK1 (S217/S221), phospho-BRAF (S446) and phospho-CRAF (S338) in a single well. This multiplex assay detects the H-Ras, N-Ras, and K-Ras isoforms for total Ras, RasG12D, and RasG12V. To confirm specific recognition of mutant Ras oncoproteins, this kit was able to detect the known KRAS G12V mutation found in the COR-L23 cell line and the NRAS G12D mutation found in THP-1 cell line, respectively. In addition, the response to BRAF inhibitors vemurafenib and dabrafenib was compared between MDA-MB-435S cells, which harbor the BRAF V600E mutation, and insensitive cell lines, using multiplex immunoassays. While COR-L23 and THP-1 cells were not responsive to vemurafenib and dabrafenib, MDA-MB-435S cells showed a dramatic reduction in pMEK1 and pERK, consistent with inhibition of mutant BRAF protein. We then analyzed commercially-sourced tissue lysates, using tumor and adjacent tissue from a patient with colon cancer harboring a KRAS G12D mutation. While multiplex protein analysis revealed a similar phosphorylation pattern for Raf and MEK proteins, the RasG12D oncoprotein was specifically observed in lysate from the tumor but not the adjacent tissue. Finally, exosomes were enriched from pancreatic cancer serum and tested using the Ras-Raf Oncoprotein panel. These exosomal preparations yielded detectable signal for Total Ras and phospho-BRAF. Our data demonstrate the utility of studying the effects of Ras oncoproteins by multiplex immunoassay in multiple sample types, including cell, tissue, and exosomal lysates. Citation Format: Joseph B. Hwang, Anthony Saporita, Qiang Xiao. Specific detection of mutant Ras oncoproteins in cell and tissue lysates by multiplex immunoassay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1453.

DHX33 is a pivotal DEAH-box RNA helicase in the multistep process of RNA polymerase I-directed transcription of the ribosomal DNA locus. We explored the regulation of DHX33 expression by Ras(V12) and ARF to determine DHX33's role in sensing these opposing signals to regulate ribosome biogenesis. In wild-type primary fibroblasts, Ras(V12) infection induced a transient increase in DHX33 protein level, as well as an rRNA transcriptional rate that was eventually suppressed by a delayed activation of the ARF/p53 pathway. DHX33 expression was exclusively controlled at the level of translation. ARF caused a dramatic reduction in polysome-associated DHX33 mRNAs, while Ras(V12) led to a complete shift of existing DHX33 mRNAs to actively translating polysomes. The translation of DHX33 by Ras(V12) was sensitive to inhibitors of phosphatidylinositol 3-kinase, mTOR, and mitogen-activated protein and was pivotal for enhanced rRNA transcription and enhanced overall cellular protein translation. In addition, DHX33 knockdown abolished Ras(V12)-induced rRNA transcription and protein translation and prevented both the in vitro and in vivo transforming properties of oncogenic Ras(V12). Our results directly implicate DHX33 as a crucial player in establishing rRNA synthesis rates in the face of Ras(V12) or ARF signals, adjusting ribosome biogenesis to match the appropriate growth or antigrowth signals.

The ARF tumor suppressor is a potent sensor of hyperproliferative cues emanating from oncogenic signaling. ARF responds to these cues by eliciting a cell cycle arrest, effectively abating the tumorigenic potential of these stimuli. Prior reports have demonstrated that oncogenic Ras(V12) signaling induces ARF through a mechanism mediated by the Dmp1 transcription factor. However, we now show that ARF protein is still induced in response to Ras(V12) in the absence of Dmp1 through the enhanced translation of existing Arf mRNAs. Here, we report that the progrowth Ras/tuberous sclerosis complex (TSC)/mTORC1 signaling pathway regulates ARF protein expression and triggers ARF-mediated tumor suppression through a novel translational mechanism. Hyperactivation of mTORC1 through Tsc1 loss resulted in a significant increase in ARF expression, activation of the p53 pathway, and a dramatic cell cycle arrest, which were completely reversed upon Arf deletion. ARF protein induced from Ras(V12) in the absence of Dmp1 repressed anchorage-independent colony formation in soft agar and tumor burden in an allograft model. Taken together, our data demonstrate the ability of the ARF tumor suppressor to respond to hypergrowth stimuli to prevent unwarranted tumor formation.

Several phosphoinositide 3-kinase (PI3K) catalytic subunit inhibitors are currently in clinical trial. We therefore sought to examine relationships between pharmacologic inhibition and somatic mutations in PI3K catalytic subunits in estrogen receptor (ER)–positive breast cancer, in which these mutations are particularly common. RNA interference (RNAi) was used to determine the effect of selective inhibition of PI3K catalytic subunits, p110α and p110β, in ER+ breast cancer cells harboring either mutation (PIK3CA) or gene amplification (PIK3CB). p110α RNAi inhibited growth and promoted apoptosis in all tested ER+ breast cancer cells under estrogen deprived-conditions, whereas p110β RNAi only affected cells harboring PIK3CB amplification. Moreover, dual p110α/p110β inhibition potentiated these effects. In addition, treatment with the clinical-grade PI3K catalytic subunit inhibitor BEZ235 also promoted apoptosis in ER+ breast cancer cells. Importantly, estradiol suppressed apoptosis induced by both gene knockdowns and BEZ235 treatment. Our results suggest that PI3K inhibitors should target both p110α and p110β catalytic subunits, whether wild-type or mutant, and be combined with endocrine therapy for maximal efficacy when treating ER+ breast cancer. [Cancer Res 2009;69(9):3955–62]

Cellular growth (i.e., macromolecular synthesis) is an essential function during the early parts of the cell cycle. For cells to transit the G1 restriction point, they must duplicate nearly their entire protein content; failure to do so would result in smaller daughter cells (12). Only recently has an emphasis been placed on the fundamental control of cell growth and its link to the cell cycle. Developments in the understanding of how the cell senses environmental nutritional cues has led to a flurry of research on understanding the mechanisms underlying growth control (40). Not surprisingly, several of these pathways converge on the synthesis of new ribosomes in the cell nucleolus and the regulation of translation. Approximately half of the cell's energy expenditure is directed toward ribosome biogenesis (26). The nucleolus, long recognized as a marker for active cellular growth, was first described in the early 1960s as the center of ribosomal DNA (rDNA) transcription and ribosome biogenesis (6, 32). This organelle is composed of three regions, on the basis of morphology at the ultrastructural level: the fibrillar centers, the dense fibrillar compartment, and the granular zone. rDNA transcription occurs in the junction region between the fibrillar centers and the surrounding dense fibrillar component, and the resulting rRNA is further processed in the periphery of the dense fibrillar component. Further posttranscriptional modifications and assembly into subunits occur in the surrounding granular region (18). While the primary mechanisms regulating these processes have been well studied in Saccharomyces cerevisiae (13), multicellular organisms demand more complex regulatory mechanisms, in that proliferative capacity is determined not only by the relative abundance of nutrients but also by complicated extracellular signals and growth factors. Indeed, previous studies have demonstrated convergence between the growth and the proliferation pathways via regulation of the tumor suppressor gene products Rb and p53 (9, 17, 43, 48). Both products are known to negatively regulate the activity of polymerase I in rDNA transcription. Oncogenes such as c-Myc also regulate the transcription of rDNA and the genes that encode ribosomal proteins, implying that an intricate network exists within the nucleolus to ensure the proper synthesis of ribosomes (7, 15, 16). The tumor suppressor p19ARF represents an attractive candidate for coupling proliferation to growth. Given its nucleolar localization (39, 44, 45) and potent induction by hyperproliferative signals (19, 20, 31, 50), ARF represents a potential nucleolar integrator of growth signals coming into the cell. It has been regarded classically as an activator of p53 through its ability to sequester Mdm2, the E3 ubiquitin ligase for p53, in the nucleolus (39, 44, 45). However, recent data have demonstrated a role for ARF in binding to and affecting the function of the ribosomal chaperone nucleophosmin (NPM), independent of its ability to regulate p53 (4, 8, 21). Furthermore, these data are consistent with those from a growing number of studies with mice and humans that describe p53-independent functions for ARF tumor suppression (35). Given ARF's nucleolar localization, its role in suppressing cellular growth and proliferation, and its ability to bind to a protein involved in ribosome biogenesis, we were inclined to explore the functional and physiological consequences of ARF disruption of growth and ribosome biogenesis. Through in vitro and in vivo assays, we utilized targeted Arf knockout mice and selective ARF knockdown via lentiviral RNA interference. Cells derived from Arf-null mice displayed significant alterations in gross nucleolar morphology and abundance and had a marked increase in basal protein synthesis levels compared to that in wild-type cells. Furthermore, this increase in protein synthesis was correlated to increased ribosome biogenesis and cytoplasmic ribosome content, implying a regulatory role for ARF in these processes. Importantly, though ARF levels are nearly undetectable in low-passage mouse embryonic fibroblasts (19), the knockdown of endogenous ARF via short hairpin RNA (shRNA) constructs mimicked the Arf-null nucleolar and ribosomal phenotype, implying an important ribosome homeostatic role for basal ARF proteins in wild-type cells. The progrowth phenotype of the Arf loss was not limited to proliferating cells, as fully differentiated osteoclasts from Arf-null mice exhibited tremendous gains in protein synthesis and overall activity in vivo. Mechanistically, all of the ribosome gains exhibited by the loss of Arf were reversed by the removal of the nucleolar NPM proto-oncogene, indicating that NPM, when untethered from ARF, promotes unrestrained ribosome biogenesis. Taken together, these data strongly argue for a moment-to-moment “thermostat”-like role for basal ARF molecules in controlling NPM-directed ribosome biogenesis and protein synthetic rates.

Androgen receptor (AR) belongs to the steroid receptor superfamily that regulates gene expression in a ligand-dependent fashion. AR is localized to the cytoplasm in the absence of androgen and translocates into the nuclei to activate gene expression in the presence of ligand. Regulation of AR nuclear import and export represents an essential step in androgen action. A nuclear localization signal (NLS) has been identified in the DNA-binding domain and hinge region of AR and other steroid receptors. Studies on nuclear export of AR, however, are limited, and what might be the underlying mechanism regulating the intracellular localization of steroid receptors is unclear. Our studies have identified a leptomycin B-insensitive nuclear export signal (NESAR) in the ligand-binding domain of AR, which is active in the absence of androgen and repressed upon ligand binding. Consistent with its androgen-sensitivity, NESAR contains amino acid residues in the immediate vicinity of the bound ligand. NESAR is necessary for AR nuclear export and is dominant over the NLS in the DNA-binding domain and hinge region in the absence of hormone. Our findings suggest that androgen can regulate NESAR and, subsequently, the NLS of the AR, providing a mechanism by which androgen regulates AR nuclear/cytoplasmic shuttling. Estrogen receptor alpha and mineralocorticoid receptor also contain functional NES, suggesting that this ligand-regulated NES is conserved among steroid receptors.

Introduction The DDX21 RNA helicase has been shown to be a nucleolar and nuclear protein involved in ribosome RNA processing and AP-1 transcription. DDX21 is highly expressed in colon cancer, lymphomas, and some breast cancers, but little is known about how DDX21 might promote tumorigenesis. Methods Immunohistochemistry was performed on a breast cancer tissue array of 187 patients. In order to study the subcellular localization of DDX21 in both tumor tissue and tumor cell lines, indirect immunofluorescence was applied. The effect of DDX21 knockdown was measured by cellular apoptosis, rRNA processing assays, soft agar growth and mouse xenograft imaging. AP-1 transcriptional activity was analyzed with a luciferase reporter and bioluminescence imaging, as well as qRT-PCR analysis of downstream target, cyclin D1, to determine the mechanism of action for DDX21 in breast tumorigenesis. Results Herein, we show that DDX21 is highly expressed in breast cancer tissues and established cell lines. A significant number of mammary tumor tissues and established breast cancer cell lines exhibit nuclear but not nucleolar localization of DDX21. The protein expression level of DDX21 correlates with cell proliferation rate and is markedly induced by EGF signaling. Mechanistically, DDX21 is required for the phosphorylation of c-Jun on Ser73 and DDX21 deficiency markedly reduces the transcriptional activity of AP-1. Additionally, DDX21 promotes rRNA processing in multiple breast cancer cell lines. Tumor cells expressing high levels of endogenous DDX21 undergo apoptosis after acute DDX21 knockdown, resulting in significant reduction of tumorigenicity in vitro and in vivo. Conclusions Our findings indicate that DDX21 expression in breast cancer cells can promote AP-1 activity and rRNA processing, and thus, promote tumorigenesis by two independent mechanisms. DDX21 could serve as a marker for a subset of breast cancer patients with higher proliferation potential and may be used as a therapeutic target for a subset of breast cancer patients. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0449-z) contains supplementary material, which is available to authorized users.

Summary: The ARF and p53 tumor suppressors are thought to act in a linear pathway to prevent cellular transformation in response to various oncogenic signals. Here, we show that loss of p53 leads to an increase in ARF protein levels, which function to limit the proliferation and tumorigenicity of p53-deficient cells by inhibiting an IFN-β-STAT1-ISG15 signaling axis. Human triple-negative breast cancer (TNBC) tumor samples with coinactivation of p53 and ARF exhibit high expression of both STAT1 and ISG15, and TNBC cell lines are sensitive to STAT1 depletion. We propose that loss of p53 function and subsequent ARF induction creates a selective pressure to inactivate ARF and propose that tumors harboring coinactivation of ARF and p53 would benefit from therapies targeted against STAT1 and ISG15 activation. : A detailed understanding of cancer cell vulnerabilities based on specific genetic and molecular signatures is imperative. In pursuit of this endeavor, Forys et al. have uncovered a proproliferative pathway that drives tumorigenesis in cells lacking the p53 and ARF tumor suppressors. They identified this oncogenic pathway as a type I interferon response mediated by IFN-β, STAT1, and ISG15. The authors further demonstrate the potential therapeutic efficacy of targeting this pathway in a subset of breast cancer patients.

DHX33 is a pivotal DEAH-box RNA helicase in the multistep process of RNA polymerase I-directed transcription of the ribosomal DNA locus. We explored the regulation of DHX33 expression by Ras(V12) and ARF to determine DHX33's role in sensing these opposing signals to regulate ribosome biogenesis. In wild-type primary fibroblasts, Ras(V12) infection induced a transient increase in DHX33 protein level, as well as an rRNA transcriptional rate that was eventually suppressed by a delayed activation of the ARF/p53 pathway. DHX33 expression was exclusively controlled at the level of translation. ARF caused a dramatic reduction in polysome-associated DHX33 mRNAs, while Ras(V12) led to a complete shift of existing DHX33 mRNAs to actively translating polysomes. The translation of DHX33 by Ras(V12) was sensitive to inhibitors of phosphatidylinositol 3-kinase, mTOR, and mitogen-activated protein and was pivotal for enhanced rRNA transcription and enhanced overall cellular protein translation. In addition, DHX33 knockdown abolished Ras(V12)-induced rRNA transcription and protein translation and prevented both the in vitro and in vivo transforming properties of oncogenic Ras(V12). Our results directly implicate DHX33 as a crucial player in establishing rRNA synthesis rates in the face of Ras(V12) or ARF signals, adjusting ribosome biogenesis to match the appropriate growth or antigrowth signals.

A novel multiplex immunoassay was developed for examining acquired resistance to Receptor Tyrosine Kinase (RTK) inhibitors. RTKs are transmembrane proteins which act as receptors for growth factors, neurotrophic factors and other extracellular signaling molecules. These cell-surface kinases are activated by extracellular ligands leading to receptor dimerization and tyrosine phosphorylation at specific residues in the cytoplasmic tails to initiate RTK-mediated signal transduction. Of the >500 known protein kinases in the human genome there are approximately 60 RTKs. They are central components of cell signaling networks and play crucial roles in normal physiological processes and disease processes ranging from diabetes to cancer. Many RTKs, such as EGFR, HER2, c-Kit, PDGFR and VEGFR, have been used as targets for drug development and RTK-targeted therapies have illustrated the utility of these treatments for selected cancers. However, in many cases, compensatory RTK signaling enables cancer cells to acquire resistance to RTK inhibitors that selectively target a single RTK. For example, some EGFR and HER2 inhibitors have led to resistance and are associated with increased expression of IGF1R. In order to understand the mechanisms of resistance to RTK inhibitors, we have developed a bead-based multiplex immunoassay capable of simultaneously detecting phosphorylation of 18 different RTKs. First we demonstrate detection of RTK phosphorylation in response to growth factor stimulation using various cell lines. Next we examined the specificity of two inhibitors targeting EGFR and HER2. These inhibitors specifically reduced growth factor-stimulated phosphorylation of EGFR and HER2, without inhibiting the activation of other RTKs. Although we did not observe compensatory activation of other RTKs in response to the two EGFR and HER2 inhibitors we tested, this study demonstrates the feasibility of using this novel 18-plex RTK panel for examining the mechanism of resistance to single RTK inhibitors. In summary, the RTK multiplex panel allowed for simultaneous detection of multiple tyrosine phosphorylated RTKs in a specific, sensitive, and reproducible manner. Citation Format: Lu Chen, Anthony J. Saporita, Wen-Rong Lie, Reeti Maheshwari, Melissa Schluter, Jehangir Mistry, Joseph Hwang. Analysis of resistance to RTK inhibitors using a novel RTK multiplex assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 205.

The serine/threonine protein kinase Akt is a key node in the PI3K pathway, one of the primary signaling cascades hyperactivated in human cancer. Emerging evidence demonstrates that the three Akt isoforms (Akt1, Akt2, and Akt3) may have unique, isoform-specific roles in key cellular processes such as differentiation and proliferation. We have developed 2-plex assays for each of the Akt isoforms in order to measure the relative levels of phospho- and total Akt1, Akt2, and Akt3. Differential regulation of Akt isoforms was characterized in multiple human cancer cell lines, including SH-SY5Y neuroblastoma cells and MCF-7 breast cancer cells. SH-SY5Y cells were used to study Akt phosphorylation in the context of differentiation. Briefly, SH-SY5Y cells were treated with retinoic acid (RA) for 3 days to induce neuronal differentiation before cells were collected, lysed, and evaluated by Luminex assay. RA-induced differentiation of SH-SY5Y cells stimulated phosphorylation of all three Akt isoforms, with Akt2 displaying the greatest induction. Multi-pathway analysis demonstrated co-induction of phospho-JNK in the RA-treated SH-SY5Y cells, consistent with the role of JNK in RA-mediated differentiation. To study differential regulation of the Akt isoforms in response to growth-stimulatory and growth-inhibitory signals, we used MCF-7 cells. In contrast to SH-SY5Y cells which express all three isoforms, MCF-7 cells only expressed Akt1 and Akt2. Serum-starved MCF-7 cells were cultured in the presence or absence of the PI3K inhibitor LY294002 prior to stimulation with insulin growth factor (IGF). Akt1 showed a greater induction of phosphorylation in response to IGF relative to Akt2. Similarly, whereas LY294002 pre-treatment reduced phospho-Akt1 to baseline levels, it only partially inhibited the IGF-dependent induction of phospho-Akt2. This suggests that Akt1 may be more sensitive to PI3K inhibition than Akt2 in certain human breast cancer cells. Collectively, our results demonstrate that Akt1, Akt2, and Akt3 are differentially regulated in human cancer cells at both the level of phosphorylation and of total protein expression. Citation Format: Anthony J. Saporita, Melissa Schluter, Debra MacIvor, Jehangir Mistry, Joseph Hwang. Interrogation of PI3K signaling via multiplex detection of differential phosphorylation of specific Akt isoforms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4611.

The p19ARF tumor suppressor limits ribosome biogenesis and responds to hyperproliferative signals to activate the p53 checkpoint response. Although its activation of p53 has been well characterized, the role of ARF in restraining nucleolar ribosome production is poorly understood. Here we report the use of a mass spectroscopic analysis to identify protein changes within the nucleoli of Arf-deficient mouse cells. Through this approach, we discovered that ARF limited the nucleolar localization of the RNA helicase DDX5, which promotes the synthesis and maturation of rRNA, ultimately increasing ribosome output and proliferation. ARF inhibited the interaction between DDX5 and nucleophosmin (NPM), preventing association of DDX5 with the rDNA promoter and nuclear pre-ribosomes. In addition, Arf-deficient cells transformed by oncogenic RasV12 were addicted to DDX5, because reduction of DDX5 was sufficient to impair RasV12-driven colony formation in soft agar and tumor growth in mice. Taken together, our findings indicate that DDX5 is a key p53-independent target of the ARF tumor suppressor and is a novel non-oncogene participant in ribosome biogenesis. Cancer Res; 71(21); 6708–17. ©2011 AACR.

One of the outstanding fundamental questions in cancer cell biology concerns how cells coordinate cellular growth (or macromolecular synthesis) with cell cycle progression and mitosis. Intuitively, rapidly dividing cells must have some control over these processes; otherwise cells would continue to shrink in volume with every passing cycle, similar to the cytoreductive divisions seen in the very early stages of embryogenesis. The problem is easily solved in unicellular organisms, such as yeast, as their growth rates are entirely dependent on nutrient availability. Multicellular organisms such as mammals, however, must have acquired additional levels of control, as nutrient availability is seldom an issue and the organism has a prodigious capacity to store necessary metabolites in the form of glycogen, lipids, and protein. Furthermore, the specific needs and specialized architecture of tissues must constrain growth for growth’s sake; if not, the necessary function of the organ could be lost. While certainly a myriad of mechanisms for preventing this exist via initiating cell death (e.g. apoptosis, autophagy, necrosis), these all depend on some external cue, such as death signals, hypoxia, lack of nutrients or survival signals. However there must also be some cell autonomous method for surveying against inappropriate growth signals (such as oncogenic stress) that occur in a stochastic fashion, possibly as a result of random mutations. The ARF tumor suppressor seems to fulfill that role, as its expression is near undetectable in normal tissues, yet is potently induced by oncogenic stress (such as overexpression of oncogenic Ras or myc). As a result of induced expression of ARF, the tumor suppressor protein p53 is stabilized and promotes cell cycle arrest. Mutations or epigenetic alterations of the INK4a/Arf locus are second only to p53 mutations in cancer cells, and in some cancers, alterations in both Arf and p53 observed, suggesting that these two tumor suppressors act coordinately to prevent unwarranted cell growth and proliferation. The aim of this review is to characterize the current knowledge in the field about both p53-dependent and independent functions of ARF as well as to summarize the present models for how ARF might control rates of cell proliferation and/or macromolecular synthesis. We will discuss potential therapeutic targets in the ARF pathway, and some preliminary attempts at enhancing or restoring the activity of this important tumor suppressor.

BACKGROUND Androgen receptor (AR) is the key molecule in androgen-refractory prostate cancer. Despite androgen ablative conditions, AR remains active and is necessary for the growth of androgen-refractory prostate cancer cells. Nuclear localization of AR is a prerequisite for its transcriptional activation. We examined AR localization in androgen-dependent and androgen-refractory prostate cancer cells. METHODS AND RESULTS We demonstrate increased nuclear localization of a GFP-tagged AR in the absence of hormone in androgen-refractory C4-2 cells compared to parental androgen-sensitive human prostate cancer LNCaP cells. Analysis of AR mutants impaired in ligand-binding indicates that the nuclear localization of AR in C4-2 cells is truly androgen-independent. The hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), inhibits basal PSA expression and disrupts the ligand-independent nuclear localization of AR at doses much lower than required to inhibit androgen-induced nuclear import. CONCLUSIONS Hsp90 is a key regulator of ligand-independent nuclear localization and activation of AR in androgen-refractory prostate cancer cells. Prostate 67: 509–520, 2007. © 2007 Wiley-Liss, Inc.

Abstract #3063 Background: Endocrine therapy with estrogen deprivation (ED) induces cell cycle arrest in sensitive estrogen receptor positive (ER+) breast cancers, but typically not apoptosis, thus relapse is common despite ED due to residual surviving tumor cells. The phosphoinositide 3-kinase (PI3K) pathway, which promotes cell survival, is hyperactivated in ER+ breast cancer due to mutations of genes in the PI3K signaling cascade, most commonly PIK3CA. We therefore investigated the role of p110α and p110β, the gene products of PIK3CA and PIK3CB, in promoting cell survival in ER+ breast cancer. Materials and Methods: RNA interference (RNAi) was used to selectively inhibit p110α and p110β expression in the ER+, PIK3CA mutant MCF-7 and T47D cell lines; the ER+, PIK3CA wild-type but PIK3CB gene amplified HCC712 cell line; and the ER-, PIK3CA and PIK3CB wild-type MDA-MB-231 cell line. Western blotting was used to confirm p110α and p110β knockdown efficiencies and to determine the effects of specific knockdowns on PI3K pathway signaling. Cell line growth was measured using a resazurin reduction assay. Apoptosis was quantified using TUNEL/Hoechst staining. Gene copy number for PIK3CB was assessed by FISH in cell lines and tumor samples. Results: PIK3CA RNAi inhibited growth and activated apoptosis in all tested ER+ cell lines under ED, whereas the addition of estrogen reversed PIK3CA RNAi-induced apoptosis. PIK3CB RNAi inhibited proliferation and promoted apoptosis in the absence of estrogen only in the PIK3CB-gene amplified HCC712 cell line. In combination, PIK3CA and PIK3CB gene knockdowns produced a synergistic apoptotic effect on T47D and HCC712 cell lines, but not in MCF-7 cells which contain low levels of p110β protein. In contrast PIK3CA and PIK3CB RNAi did not promote apoptosis in the ER- MDA-MB-231 cell line. Finally, since PIK3CB gene amplification has not been described previously we conducted a screen by FISH and array CGH and detected low-level PIK3CB copy number gain or amplification in ∼5% of clinical samples from a series of breast cancer patients representing diverse clinical phenotypes (ER+, ER-, ERBB2+). Conclusions: Inhibition of the PI3K pathway through suppression of PI3K catalytic subunit expression produces a synthetic lethal effect in the absence of estrogen specifically in ER+ breast cancer cells. These data establish a preclinical rationale for combining endocrine therapy with PI3K pharmacological inhibitors in ER+ breast cancer, but suggest that both PIK3CA and PIK3CB are therapeutic targets and gene mutation or amplification in both catalytic subunits may have to be taken into account when identiying a sensitive population for PI3 kinase-directed therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3063.