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1.
Cancer Res Commun ; 3(7): 1366-1377, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37501682

RESUMEN

NF1 is a key tumor suppressor that represses both RAS and estrogen receptor-α (ER) signaling in breast cancer. Blocking both pathways by fulvestrant (F), a selective ER degrader, together with binimetinib (B), a MEK inhibitor, promotes tumor regression in NF1-depleted ER+ models. We aimed to establish approaches to determine how NF1 protein levels impact B+F treatment response to improve our ability to identify B+F sensitive tumors. We examined a panel of ER+ patient-derived xenograft (PDX) models by DNA and mRNA sequencing and found that more than half of these models carried an NF1 shallow deletion and generally have low mRNA levels. Consistent with RAS and ER activation, RET and MEK levels in NF1-depleted tumors were elevated when profiled by mass spectrometry (MS) after kinase inhibitor bead pulldown. MS showed that NF1 can also directly and selectively bind to palbociclib-conjugated beads, aiding quantification. An IHC assay was also established to measure NF1, but the MS-based approach was more quantitative. Combined IHC and MS analysis defined a threshold of NF1 protein loss in ER+ breast PDX, below which tumors regressed upon treatment with B+F. These results suggest that we now have a MS-verified NF1 IHC assay that can be used for patient selection as a complement to somatic genomic analysis. Significance: A major challenge for targeting the consequence of tumor suppressor disruption is the accurate assessment of protein functional inactivation. NF1 can repress both RAS and ER signaling, and a ComboMATCH trial is underway to treat the patients with binimetinib and fulvestrant. Herein we report a MS-verified NF1 IHC assay that can determine a threshold for NF1 loss to predict treatment response. These approaches may be used to identify and expand the eligible patient population.


Asunto(s)
Neoplasias de la Mama , Proteogenómica , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neurofibromina 1/genética , Fulvestrant/farmacología , Receptores de Estrógenos/genética , Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción NFI , ARN Mensajero , Quinasas de Proteína Quinasa Activadas por Mitógenos
2.
Breast Cancer Res ; 24(1): 68, 2022 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-36258226

RESUMEN

BACKGROUND: Ductal carcinoma in situ (DCIS) is the most common type of in situ premalignant breast cancers. What drives DCIS to invasive breast cancer is unclear. Basal-like invasive breast cancers are aggressive. We have previously shown that NRAS is highly expressed selectively in basal-like subtypes of invasive breast cancers and can promote their growth and progression. In this study, we investigated whether NRAS expression at the DCIS stage can control transition from luminal DCIS to basal-like invasive breast cancers. METHODS: Wilcoxon rank-sum test was performed to assess expression of NRAS in DCIS compared to invasive breast tumors in patients. NRAS mRNA levels were also determined by fluorescence in situ hybridization in patient tumor microarrays (TMAs) with concurrent normal, DCIS, and invasive breast cancer, and association of NRAS mRNA levels with DCIS and invasive breast cancer was assessed by paired Wilcoxon signed-rank test. Pearson's correlation was calculated between NRAS mRNA levels and basal biomarkers in the TMAs, as well as in patient datasets. RNA-seq data were generated in cell lines, and unsupervised hierarchical clustering was performed after combining with RNA-seq data from a previously published patient cohort. RESULTS: Invasive breast cancers showed higher NRAS mRNA levels compared to DCIS samples. These NRAShigh lesions were also enriched with basal-like features, such as basal gene expression signatures, lower ER, and higher p53 protein and Ki67 levels. We have shown previously that NRAS drives aggressive features in DCIS-like and basal-like SUM102PT cells. Here, we found that NRAS-silencing induced a shift to a luminal gene expression pattern. Conversely, NRAS overexpression in the luminal DCIS SUM225 cells induced a basal-like gene expression pattern, as well as an epithelial-to-mesenchymal transition signature. Furthermore, these cells formed disorganized mammospheres containing cell masses with an apparent reduction in adhesion. CONCLUSIONS: These data suggest that elevated NRAS levels in DCIS are not only a marker but can also control the emergence of basal-like features leading to more aggressive tumor activity, thus supporting the therapeutic hypothesis that targeting NRAS and/or downstream pathways may block disease progression for a subset of DCIS patients with high NRAS.


Asunto(s)
Neoplasias de la Mama , Carcinoma Ductal de Mama , Carcinoma Intraductal no Infiltrante , Humanos , Femenino , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/patología , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Carcinoma Ductal de Mama/patología , Proteína p53 Supresora de Tumor/genética , Neoplasias de la Mama/patología , Hibridación Fluorescente in Situ , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , ARN Mensajero , Progresión de la Enfermedad , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo
3.
Cancer Cell ; 37(3): 387-402.e7, 2020 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-32142667

RESUMEN

We report that neurofibromin, a tumor suppressor and Ras-GAP (GTPase-activating protein), is also an estrogen receptor-α (ER) transcriptional co-repressor through leucine/isoleucine-rich motifs that are functionally independent of GAP activity. GAP activity, in turn, does not affect ER binding. Consequently, neurofibromin depletion causes estradiol hypersensitivity and tamoxifen agonism, explaining the poor prognosis associated with neurofibromin loss in endocrine therapy-treated ER+ breast cancer. Neurofibromin-deficient ER+ breast cancer cells initially retain sensitivity to selective ER degraders (SERDs). However, Ras activation does play a role in acquired SERD resistance, which can be reversed upon MEK inhibitor addition, and SERD/MEK inhibitor combinations induce tumor regression. Thus, neurofibromin is a dual repressor for both Ras and ER signaling, and co-targeting may treat neurofibromin-deficient ER+ breast tumors.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Neurofibromina 1/genética , Secuencias de Aminoácidos , Animales , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proteínas Co-Represoras , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Células MCF-7 , Ratones Desnudos , Ratones SCID , Mutación , Neurofibromina 1/química , Neurofibromina 1/metabolismo , Transducción de Señal , Tamoxifeno/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/metabolismo
4.
Sci Rep ; 8(1): 16932, 2018 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-30446677

RESUMEN

Ras GTPases are powerful drivers for tumorigenesis, but directly targeting Ras for treating cancer remains challenging. The growth and transforming activity of the aggressive basal-like breast cancer (BLBC) are driven by N-Ras. To target N-Ras in BLBC, this study screened existing pharmacologically active compounds for the new ability to induce N-Ras degradation, which led to the identification of flunarizine (FLN), previously approved for treating migraine and epilepsy. The FLN-induced N-Ras degradation was not affected by a 26S-proteasome inhibitor. Rather, it was blocked by autophagy inhibitors. Furthermore, N-Ras can be seen co-localized with active autophagosomes upon FLN treatment, suggesting that FLN alters the autophagy pathway to degrade N-Ras. Importantly, FLN treatment recapitulated the effect of N-RAS silencing in vitro by selectively inhibiting the growth of BLBC cells, but not that of breast cancer cells of other subtypes. In addition, in vivo FLN inhibited tumor growth of a BLBC xenograft model. In conclusion, this proof-of-principle study presents evidence that the autophagy pathway can be coerced by small molecule inhibitors, such as FLN, to degrade Ras as a strategy to treat cancer. FLN has low toxicity and should be further investigated to enrich the toolbox of cancer therapeutics.


Asunto(s)
Autofagia/efectos de los fármacos , Flunarizina/farmacología , Proteínas ras/metabolismo , Animales , Autofagosomas , Autofagia/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Genes Reporteros , Humanos , Ratones , Proteolisis , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genética
5.
Cell Rep ; 12(3): 511-24, 2015 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-26166574

RESUMEN

Basal-like breast cancers (BLBCs) are aggressive, and their drivers are unclear. We have found that wild-type N-RAS is overexpressed in BLBCs but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, whereas overexpression enhances these processes even in preinvasive BLBC cells. We identified N-Ras-responsive genes, most of which encode chemokines; e.g., IL8. Expression levels of these chemokines and N-RAS in tumors correlate with outcome. N-Ras, but not K-Ras, induces IL-8 by binding and activating the cytoplasmic pool of JAK2; IL-8 then acts on both the cancer cells and stromal fibroblasts. Thus, BLBC progression is promoted by increasing activities of wild-type N-Ras, which mediates autocrine/paracrine signaling that can influence both cancer and stroma cells.


Asunto(s)
Neoplasias de la Mama/metabolismo , GTP Fosfohidrolasas/metabolismo , Interleucina-8/metabolismo , Janus Quinasa 2/metabolismo , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , GTP Fosfohidrolasas/genética , Genes ras , Humanos , Janus Quinasa 2/genética , Ratones , Ratones Desnudos , Ratones Transgénicos , Transducción de Señal
6.
Cell Biosci ; 5: 10, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25774287

RESUMEN

BACKGROUND: The INT6 gene was first discovered as a site of integration in mouse mammary tumors by the mouse mammary tumor virus; however, INT6's role in the development of human breast cancer remains largely unknown. By gene silencing, we have previously shown that repressing INT6 promotes transforming activity in untransformed human mammary epithelial cells. In the present study, guided by microarray data of human tumors, we have discovered a role of Int6 in stromal fibroblasts. RESULTS: We searched microarray databases of human tumors to assess Int6's role in breast cancer. While INT6 expression levels, as expected, were lower in breast tumors than in adjacent normal breast tissue samples, INT6 expression levels were also substantially lower in tumor stroma. By immunohistochemistry, we determined that the low levels of INT6 mRNA observed in the microarray databases most likely occurs in stromal fibroblasts, because far fewer fibroblasts in the tumor tissue showed detectable levels of the Int6 protein. To directly investigate the effects of Int6 repression on fibroblasts, we silenced INT6 expression in immortalized human mammary fibroblasts (HMFs). When these INT6-repressed HMFs were co-cultured with breast cancer cells, the abilities of the latter to form colonies in soft agar and to invade were enhanced. We analyzed INT6-repressed HMFs and found an increase in the levels of a key carcinoma-associated fibroblast (CAF) marker, smooth muscle actin. Furthermore, like CAFs, these INT6-repressed HMFs secreted more stromal cell-derived factor 1 (SDF-1), and the addition of an SDF-1 antagonist attenuated the INT6-repressed HMFs' ability to enhance soft agar colony formation when co-cultured with cancer cells. These INT6-repressed HMFs also expressed high levels of mesenchymal markers such as vimentin and N-cadherin. Intriguingly, when mesenchymal stem cells (MSCs) were induced to form CAFs, Int6 levels were reduced. CONCLUSION: These data suggest that besides enhancing transforming activity in epithelial cells, INT6 repression can also induce fibroblasts, and possibly MSCs as well, via mesenchymal-mesenchymal transitions to promote the formation of CAFs, leading to a proinvasive microenvironment for tumorigenesis.

7.
Methods Enzymol ; 535: 25-38, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24377915

RESUMEN

While Ras GTPases are best known for mediating growth factor signaling on the plasma membrane, these proteins also have surprisingly complex activities in the endosome. Assisted by a method called bimolecular fluorescent complementation (BiFC), which can detect weak and transient protein-protein interactions and reveal where the binding takes place in live cells, we have identified three effectors, Cdc42, CHMP6, and VPS4A that interact with Ras proteins in endosomes. These effectors are all necessary for Ras-induced transformation, suggesting that for Ras proteins to efficiently induce tumor formation, they must also activate effectors in cytoplasm, such as those in endosomes. Here, we describe how BiFC can be used to detect and screen for Ras effectors and for readily revealing where in the cell the binding occurs.


Asunto(s)
Endosomas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Transducción de Señal , Proteínas ras/metabolismo , Animales , Proteínas Bacterianas/biosíntesis , Línea Celular Tumoral , Biblioteca de Genes , Células HEK293 , Humanos , Proteínas Luminiscentes/biosíntesis , Ratones , Microscopía Fluorescente , Células 3T3 NIH , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis
8.
Mol Cell Biol ; 34(3): 374-85, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24248599

RESUMEN

Ras can act on the plasma membrane (PM) to mediate extracellular signaling and tumorigenesis. To identify key components controlling Ras PM localization, we performed an unbiased screen to seek Schizosaccharomyces pombe mutants with reduced PM Ras. Five mutants were found with mutations affecting the same gene, S. pombe erf2 (sp-erf2), encoding sp-Erf2, a palmitoyltransferase, with various activities. sp-Erf2 localizes to the trans-Golgi compartment, a process which is mediated by its third transmembrane domain and the Erf4 cofactor. In fission yeast, the human ortholog zDHHC9 rescues the phenotypes of sp-erf2 null cells. In contrast, expressing zDHHC14, another sp-Erf2-like human protein, did not rescue Ras1 mislocalization in these cells. Importantly, ZDHHC9 is widely overexpressed in cancers. Overexpressing ZDHHC9 promotes, while repressing it diminishes, Ras PM localization and transformation of mammalian cells. These data strongly demonstrate that sp-Erf2/zDHHC9 palmitoylates Ras proteins in a highly selective manner in the trans-Golgi compartment to facilitate PM targeting via the trans-Golgi network, a role that is most certainly critical for Ras-driven tumorigenesis.


Asunto(s)
Aciltransferasas/metabolismo , Membrana Celular/metabolismo , Transformación Celular Neoplásica , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas ras/metabolismo , Aciltransferasas/genética , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Secuencia Conservada/genética , Evolución Molecular , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Microscopía Confocal , Mutación , Células 3T3 NIH , Ácidos Palmíticos/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas ras/genética , Red trans-Golgi/metabolismo
9.
Small GTPases ; 3(4): 236-9, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22735486

RESUMEN

Ras proteins are best known to function on the plasma membrane to mediate growth factor signaling. Controlling the length of time that Ras proteins stay on the plasma membrane is an effective way to properly modulate the intensity and duration of growth factor signaling. It has been shown previously that H- and N-Ras proteins in the GTP-bound state can be ubiquitylated via a K-63 linkage, which leads to endosome internalization and results in a negative-feedback loop for efficient signal attenuation. In a more recent study, two new Ras effectors have been isolated, CHMP6 and VPS4A, which are components of the ESCRT-III complex, best known for mediating protein sorting in the endosomes. Surprisingly, these molecules are required for efficient Ras-induced transformation. They apparently do so by controlling recycling of components of the Ras pathway back to the plasma membrane, thus creating a positive-feedback loop to enhance growth factor signaling. These results suggest the fates of endosomal Ras proteins are complex and dynamic - they can be either stored and/or destroyed or recycled. Further work is needed to decipher how the fate of these endosomal Ras proteins is determined.


Asunto(s)
Membrana Celular/metabolismo , Proteínas ras/metabolismo , Animales , Citoplasma/metabolismo , Endosomas/metabolismo , Humanos
10.
Cancer Lett ; 268(2): 202-11, 2008 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-18477500

RESUMEN

We have reported that the ectopic expression of progesterone receptor (PR) in MCF-7 cells disrupted the effects of estradiol-17beta (E2), and this anti-estrogenic effect of PR was associated with heightened E2 metabolism and inhibition of estrogen receptor (ER) binding to estrogen response element (ERE). This study determined if the transfected PR was also able to abolish the effect of other estrogens including estrone (E1), estriol (E3) and estradiol-17alpha. The study revealed that the transfected PR in MCF-7 cells abolished the growth-stimulatory effects of E1 and E2, but had minimal effect on the effects of E3 and estradiol-17alpha. This observation is associated with a faster metabolic inactivation of E1 and E2 than that of E3 and estradiol-17alpha. The conditioned media collected after 72h of E3 treatment from PR-transfected MCF-7 cells retained full estrogenic potential whereas E2-treated conditioned media after 72h have lost all of the estrogenic activity. On the other hand, PR was able to inhibit ER-ERE binding induced by all the estrogens. This suggests that the estrogen-selective anti-estrogenic effect of transfected PR was due to the difference in rate of metabolic inactivation of different estrogens in MCF-7 cells. It is plausible that inhibition of ER-ERE interaction by PR resulted in faster estrogen-ER dissociation, and the dissociated E1 and E2 were metabolically inactivated but enzymes for E3 and estradiol-17alpha inactivation were lacking in MCF-7 cells. The findings suggest an interesting mechanism by which the disruption of ER-ERE interaction heightens the inactivation of estrogens in breast cancer cells.


Asunto(s)
Neoplasias de la Mama/patología , Moduladores de los Receptores de Estrógeno/farmacología , Receptores de Progesterona/fisiología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Receptores de Estrógenos/metabolismo , Elementos de Respuesta , Transfección
11.
Breast Cancer Res Treat ; 110(1): 111-25, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17687644

RESUMEN

Over half of breast cancer cases are estrogen-dependent and strategies to combat estrogen-dependent breast cancer have been to either block the activation of estrogen receptor (ER) or diminish the supply of estrogens. Our previous work documented that estrogen-independent expression of progesterone receptor (PR) in MCF-7 cells markedly disrupted the effects of estrogen. In this study, we have developed an adenovirus-mediated gene delivery system to study the specific involvement of PR isoform A (PR-A) and PR-B in the anti-estrogenic effect and its mechanism of action. The results revealed that PR-B, but not PR-A, exhibited distinct anti-estrogenic effect on E2-induced cell growth, gene expression, and ER-ERE interaction in a ligand-independent manner. The anti-estrogenic effect of PR-B was also associated with heightened metabolism and increased cellular uptake of estradiol-17 beta (E2). We have also found that the B-upstream segment of PR-B alone was able to inhibit E2-induced ER-ERE interaction and cellular uptake of E2. Although PR-A alone did not affect E2-induced ER activity, it antagonized the anti-estrogenic effect of PR-B in a concentration-dependent manner. The findings suggest an important mechanism of maintaining a favorable level of ER activity by PR-A and PR-B in estrogen target cells for optimal growth and differentiation. The potential anti-estrogenic mechanism of PR-B may be exploited for breast cancer therapy.


Asunto(s)
Neoplasias de la Mama/patología , Antagonistas de Estrógenos , Receptores de Progesterona/fisiología , Adenoviridae/genética , Secuencias de Aminoácidos , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Proliferación Celular , Estradiol/metabolismo , Estradiol/farmacología , Femenino , Terapia Genética , Humanos , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/química , Receptores de Progesterona/genética , Elementos de Respuesta
12.
J Biol Chem ; 280(17): 17480-7, 2005 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-15728178

RESUMEN

The expression of progesterone receptor (PR) is normally estrogen-dependent, and progesterone is only active in target cells following estrogen exposure. This study revealed that the effect of estrogen was markedly disrupted by estrogen-independent expression of PR. Transfection of PR in estrogen receptor (ER)-positive MCF-7 cells abolished the estradiol-17beta growth stimulatory effect that was observed in the parental cells and the vector-transfected controls in a ligand-independent manner. The antiestrogenic effect was also observed at the level of gene transcription. Estradiol-17beta (E2)-induced gene expression of pS2 and GREB1 was impaired by 50-75% after 24-72 h of E2 treatment in PR-transfected cells. Promoter interference assay revealed that PR transfection drastically inhibited E2-mediated ER binding to estrogen response elements (ERE). The antiestrogenic effects of transfected PR are associated with enhanced metabolism of E2. HPLC analysis of [3H]E2 in the samples indicated that the percentage of [3H]E2 metabolized by PR-transfected cells in 6 h is similar to that by vector-transfected control cells in 24 h (77 and 80%, respectively). The increased metabolism of E2 may, in turn, be caused by increased cellular uptake of E2, as demonstrated by whole cell binding of [3H]E2. The findings open up a new window for a hitherto unknown functional relationship between the PR and ER. The antiestrogenic effect of transfected PR also provides a potential therapeutic strategy for estrogen-dependent breast cancer.


Asunto(s)
Neoplasias de la Mama/metabolismo , Moduladores de los Receptores de Estrógeno/metabolismo , Receptores de Progesterona/metabolismo , Western Blotting , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Cromatografía Líquida de Alta Presión , Estradiol/metabolismo , Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Regulación de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción Genética , Transfección
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