Jillian M. Silva

Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3.

If neuropilin-2 and the growth factor VEGF-C don’t come together, lymphatic vessels don’t branch apart.

Inhibition of mitochondrial function induces an integrated stress response in oligodendroglia.

Maternal inheritance of a pathogenic point mutation within complex I of the mitochondrial genome causes Lebers hereditary optic neuropathy (LHON), resulting in the neurodegeneration and demyelination of the optic nerve. The integrated stress response (ISR), a signaling pathway that responds to various stresses by activating a common set of genes, has been linked to both mitochondrial defects and demyelinating diseases. Therefore, we wanted to determine whether mitochondrial dysfunction induced by complex I inhibition with rotenone can activate the ISR, specifically by the ER kinase PERK, in oligodendroglial cells. Our complex I-deficient oligodendroglial model reproduced similar biochemical defects as in LHON by decreasing ATP synthesis and ATP levels. The same doses of rotenone that reduced ATP production also induced dose-dependent increases in PERK and eIF2alpha phosphorylation as well as activated the ISR stress genes, ATF4 and CHOP. In addition, complex I inhibition at these same concentrations induced a PERK-dependent activation of the cell death kinase, JNK, and inhibited oligodendroglial proliferation. Taken together, our results demonstrate that activation of the ISR may be one example of mitochondrial retrograde signaling in response to complex I deficiency and we suggest that this response mechanism may be relevant to the pathophysiology of LHON.

Activating BRAF and PIK3CA Mutations Cooperate to Promote Anaplastic Thyroid Carcinogenesis

Thyroid malignancies are the most common type of endocrine tumors. Of the various histologic subtypes, anaplastic thyroid carcinoma (ATC) represents a subset of all cases but is responsible for a significant proportion of thyroid cancer-related mortality. Indeed, ATC is regarded as one of the more aggressive and hard to treat forms of cancer. To date, there is a paucity of relevant model systems to critically evaluate how the signature genetic abnormalities detected in human ATC contribute to disease pathogenesis. Mutational activation of the BRAF protooncogene is detected in approximately 40% of papillary thyroid carcinoma (PTC) and in 25% of ATC. Moreover, in ATC, mutated BRAF is frequently found in combination with gain-of-function mutations in the p110 catalytic subunit of PI3′-Kinase (PIK3CA) or loss-of-function alterations in either the p53 (TP53) or PTEN tumor suppressors. Using mice with conditional, thyrocyte-specific expression of BRAFV600E, we previously developed a model of PTC. However, as in humans, BRAFV600E-induced mouse PTC is indolent and does not lead to rapid development of end-stage disease. Here, we use mice carrying a conditional allele of PIK3CA to demonstrate that, although mutationally activated PIK3CAH1047R is unable to drive transformation on its own, when combined with BRAFV600E in thyrocytes, this leads to development of lethal ATC in mice. Combined, these data demonstrate that the BRAFV600E cooperates with either PIK3CAH1074R or with silencing of the tumor-suppressor PTEN, to promote development of anaplastic thyroid carcinoma. Implications: This genetically relevant mouse model of ATC will be an invaluable platform for preclinical testing of pathway-targeted therapies for the prevention and treatment of thyroid carcinoma. Mol Cancer Res; 12(7); 979–86. ©2014 AACR.

BRAFV600E Cooperates with PI3K Signaling, Independent of AKT, to Regulate Melanoma Cell Proliferation

Mutationally activated BRAFV600E cooperates with PTEN silencing in the conversion of normal melanocytes to metastatic melanoma cells, but the mechanism underlying this cooperation is poorly understood. Here, the consequences of pharmacologic blockade of BRAFV600E or phosphoinositide 3-kinase (PI3K) signaling were explored using pathway-targeted inhibitors and a panel of human BRAF-mutated melanoma-derived cell lines. Blockade of BRAFV600E → MEK1/2 → ERK1/2 or class I PI3K inhibited melanoma proliferation, whereas inhibition of AKT had only modest effects, even in cells with mutated or amplified AKT. Although single-agent inhibition of either BRAFV600E or PI3K signaling elicited antiproliferative effects, combinatorial inhibition was more potent. Analysis of signaling downstream of BRAFV600E or PI3K revealed that these pathways cooperated to regulate protein synthesis through AKT-independent, mTOR complex 1 (mTORC1)-dependent effects on p70S6K, ribosomal protein S6, and 4E-BP1 phosphorylation. Moreover, inhibition of mTORC1/2 inhibited cell proliferation as profoundly as single-agent inhibition of either BRAFV600E or PI3K signaling. These data reveal a mechanism by which BRAFV600E and PI3K signaling cooperate to regulate melanoma proliferation through AKT-independent effects on protein translation. Furthermore, this study provides a potential foundation for pathway-targeted combination therapy designed to enhance the therapeutic benefit to patients with melanoma that contain combined alterations in BRAF and PI3K signaling. Implications: PI3K, but not AKT, represent potential targets for melanoma therapy. Mol Cancer Res; 12(3); 447–63. ©2014 AACR.

Expression and activity of poly(ADP-ribose) glycohydrolase in cultured astrocytes, neurons, and C6 glioma cells.

Poly(ADP-ribose) metabolism plays a major role in DNA repair, transcription, replication, and recombination. Poly(ADP-ribose) polymerases are localized primarily to the nucleus, whereas significant levels of poly(ADP-ribose) glycohydrolase (PARG) are believed to be located in the cytoplasm. Only one PARG gene has been identified, but prior studies have reported multiple products of this gene. Here we studied PARG activity and PARG gene expression in several CNS cell types that span the cell growth spectrum: rapidly dividing C6 glioma tumor cells, dividing astrocytes, non-dividing astrocytes (due to contact inhibition), and post-mitotic neurons. Activity assays showed no overall differences between these cell types, but the nuclear to cytoplasmic ratio of PARG activity was highest in C6 glioma cells and lowest in neurons. Western blotting revealed full-length PARG as well as lower molecular weight PARG species in all four cell types.

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

PIK3CA-mutated melanoma cells rely on cooperative signaling through mTORC1/2 for sustained proliferation

Malignant conversion of BRAF‐ or NRAS‐mutated melanocytes into melanoma cells can be promoted by PI3′‐lipid signaling. However, the mechanism by which PI3′‐lipid signaling cooperates with mutationally activated BRAF or NRAS has not been adequately explored. Using human NRAS‐ or BRAF‐mutated melanoma cells that co‐express mutationally activated PIK3CA, we explored the contribution of PI3′‐lipid signaling to cell proliferation. Despite mutational activation of PIK3CA, melanoma cells were more sensitive to the biochemical and antiproliferative effects of broader spectrum PI3K inhibitors than to an α‐selective PI3K inhibitor. Combined pharmacological inhibition of MEK1/2 and PI3K signaling elicited more potent antiproliferative effects and greater inhibition of the cell division cycle compared to single‐agent inhibition of either pathway alone. Analysis of signaling downstream of MEK1/2 or PI3K revealed that these pathways cooperate to regulate cell proliferation through mTORC1‐mediated effects on ribosomal protein S6 and 4E‐BP1 phosphorylation in an AKT‐dependent manner. Although PI3K inhibition resulted in cytostatic effects on xenografted NRASQ61H/PIK3CAH1047R melanoma, combined inhibition of MEK1/2 plus PI3K elicited significant melanoma regression. This study provides insights as to how mutationally activated PIK3CA acts in concert with MEK1/2 signaling to cooperatively regulate mTORC1/2 to sustain PIK3CA‐mutated melanoma proliferation.

Depression of cell-mediated immunity in plasmacytoma-bearing mice

Previous reports have documented that mice bearing plasmacytomas (PC) are suppressed in their ability to mount a primary antibody response, whereas cellular immunity appears to be normal. Studies presented here provide evidence that T cell responsiveness is also depressed in BALB/c mice bearing the Lieberman plasmacytoma (Lpc‐1). For instance, splenocytes from mice bearing large tumours were impaired in their in vitro ability to respond to the T cell mitogen, mount an appropriate alloreactive cytolytic T lymphocyte response, and produce interleukin‐2 (IL‐2). A population of suppressor cells was detected in the spleens 7 days after tumour implantation as evidenced by their ability to prevent normal splenocytes not only from responding to antigens in mixed lymphocyte culture, but also from producing IL‐2. A similar inhibitory effect was observed with culture supernatants of these cells, indicating the existence of a soluble suppressive factor. Therefore, the present study demonstrates that cellular immune responses are impaired in mice bearing Lpc‐1 tumours and that this effect may be due to the generation of suppressor cells and/or a suppressive factor.

Abstract 1370: EGFR-mediated Spred1 phosphorylation inhibits NF1 to sustain constitutive Ras/MAPK signaling

Spred proteins negatively regulate Ras/MAPK signaling following growth factor stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. Spred1 and NF1 loss-of-function mutations occur across multiple cancer types including non-small cell lung carcinoma, glioblastoma, melanoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that EGFR signaling disrupts Spred1-NF1 binding. Mass spectrometry was performed on cells overexpressing EGFRL858R to identify potential phosphorylation sites on Spred1 and NF1 that could disrupt Spred1-NF1 binding by steric hindrance. A serine phosphorylation site on Spred1 was identified in which a phosphomimetic and phosphodeficient mutant decreased or increased Spred1-NF1 binding, respectively. Phosphomimetic Spred1 is unable to suppress Ras-GTP following EGF stimulation. Therefore, phosphorylation of Spred1 at this site by a serine kinase downstream of EGFR may disrupt Spred1-NF1 binding. To identify the Spred1 kinase we are performing an in vitro kinase assay and an unbiased CRISPRa screen. Our findings provide one potential mechanism by which EGFR signaling disrupts negative feedback to sustain constitutive Ras signaling. Furthermore, this work may elucidate a novel therapeutic target for restoring NF1-mediated inhibition of Ras. Citation Format: Evan Markegard, Ellen L. Mercado, Jillian M. Silva, Jacqueline Galeas, Marena I. Trinidad, Anatoly Urisman, Frank McCormick. EGFR-mediated Spred1 phosphorylation inhibits NF1 to sustain constitutive Ras/MAPK signaling [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 1370. doi:10.1158/1538-7445.AM2017-1370

Abstract 29: MEK and PI3K signaling cooperate through mTORC1/2 to promote PIK3CA mutant melanoma cell proliferation

Oncogenic transformation of mutationally activated BRAF or NRAS melanocytes often requires the cooperation of genetic aberrations in the phosphoinositide 3-kinase (PI3K) pathway to promote melanomagenesis. Activating point mutations in the p110α catalytic subunit of PI3K are detected at a low frequency in both BRAF and NRAS mutant melanomas, yet the principle mechanism of this cooperation remains elusive. Thus, using BRAFV600E/PIK3CAH1047R, NRASQ61H/PIK3CAH1047R, and PIK3CAE545K mutant melanoma cells derived from metastatic melanoma patients treated with pathway-targeted inhibitors, we examined the contribution of mutational activation of PIK3CA to melanoma maintenance and signaling and the consequential response of these PIK3CA mutant cells to α-specific PI3K inhibition. Combined MEK and isoform-selective PI3K inhibition elicited more potent anti-proliferative effects and greater suppression of S-phase progression of the cell cycle compared to single-agent inhibition of either pathway. Analysis of signaling downstream of MEK or PI3K revealed that these pathways cooperated to regulate PIK3CA cell proliferation through mTORC1-mediated effects on ribosomal protein S6 and 4E-BP1 phosphorylation in an AKT-dependent manner. Despite the profound anti-proliferative and biochemical effects of α-specific or PI3Kβ-sparing class I PI3K inhibition in vitro, these agents elicited largely cytostatic effects on PIK3CA xenograft tumors. However, combined inhibition of PI3Kβ-sparing class I PI3K and MEK did significantly cooperate to reduce tumor growth compared to the corresponding monotherapies. Furthermore, this study provides a biochemical mechanism to explain how mTORC1/2 moderates the cooperation of MEK and PI3K signaling for the maintenance of PIK3CA mutant melanoma cell proliferation. Citation Format: Jillian M. Silva, Marian M. Deuker, Bruce C. Baguley, Martin McMahon. MEK and PI3K signaling cooperate through mTORC1/2 to promote PIK3CA mutant melanoma cell proliferation. [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 29.