Inhibition of SHP2 in basal-like and triple-negative breast cells induces basal-to-luminal transition, hormone dependency, and sensitivity to anti-hormone treatment

Background The Src homology phosphotyrosyl phosphatase 2 (SHP2) is a positive effector of cell growth and survival signaling as well transformation induced by multiple tyrosine kinase oncogenes. Since the basal-like and triple-negative breast cancer (BTBC) is characterized by dysregulation of multiple tyrosine kinase oncogenes, we wanted to determine the importance of SHP2 in BTBC cell lines. Methods Short hairpin RNA-based and dominant-negative expression-based SHP2 inhibition techniques were used to interrogate the functional importance of SHP2 in BTBC cell biology. In addition, cell viability and proliferation assays were used to determine hormone dependency for growth and sensitivity to anti-estrogen treatment. Results We show that inhibition of SHP2 in BTBC cells induces luminal-like epithelial morphology while suppressing the mesenchymal and invasive property. We have termed this process as basal-to-luminal transition (BLT). The occurrence of BLT was confirmed by the loss of the basal marker alpha smooth muscle actin and the acquisition of the luminal marker cytokeratin 18 (CK18) expression. Furthermore, the occurrence of BLT led to estrogen receptor alpha (ERα) expression, hormone dependency, and sensitivity to tamoxifen treatment. Conclusions Our data show that inhibition of SHP2 induces BLT, ERα expression, dependency on estrogen for growth, and sensitivity to anti-hormone therapy. Therefore, inhibition of SHP2 may provide a therapeutic benefit in basal-like and triple-negative breast cancer

SHP2 Acts Both Upstream and Downstream of Multiple Receptor Tyrosine Kinases to Promote Basal-like and Triple-Negative Breast Cancer

Introduction: Dysregulated receptor tyrosine kinase (RTK) signaling is a common occurrence in basal-like and triple-negative breast cancer (BTBC). As a result, RTK-targeting therapies have been initiated but proved difficult, mainly owing to the multiplicity of dysregulated RTKs. Hence, targeting master regulators of RTK signaling might alleviate this obstacle. Before that, however, defining the mechanism of such molecules is required. In this report, we show that the Src homology phosphotyrosyl phosphatase 2 (SHP2) is a master regulator of RTK expression and signaling in BTBC. Methods: Xenograft tumor growth studies were used to determine the effect of SHP2 inhibition on tumorigenesis and/or metastasis. Cell proliferation rate, anchorage-independent growth, mammosphere formation, and ALDEFLUOR assays were used to compare the relative functional importance of SHP2 and the epidermal growth factor receptor (EGFR) in BTBC cells. Immunohistochemistry and immunofluorescence analyses were used to determine the state of SHP2 and EGFR coexpression in BTBC. Analysis of mitogenic and cell survival signaling was performed to show SHP2’s role in signaling by multiple RTKs. Results: Inhibition of SHP2 in BTBC cells suppresses their tumorigenic and metastatic properties. Because EGFR is the most commonly dysregulated RTK in BTBC, we first tested the effect of SHP2 inhibition on EGFR signaling and found that SHP2 is important not only for mediation of the Ras/extracellular signal-regulated kinase and the phosphatidyl inositol 3-kinase/Akt signaling pathways but also for the expression of the receptor itself. The existence of a tight association between SHP2 and EGFR expression in tumors and cell lines further suggested the importance of SHP2 in EGFR expression. Comparison of relative biological significance showed the superiority of SHP2 inhibition over that of EGFR, suggesting the existence of additional RTKs regulated by SHP2. Indeed, we found that the expression as well as the signaling efficiency of c-Met and fibroblast growth factor receptor 1, two other RTKs known to be dysregulated in BTBC, are SHP2-dependent. To our knowledge, this is the first demonstration of SHP2 acting both upstream and downstream of RTKs to promote signaling. Conclusions: SHP2 upregulates the expression and signaling of multiple RTKs to promote BTBC. These findings provide a mechanistic explanation for the superiority of SHP2 inhibition in BTBC

The NANOGrav 15 yr Data Set: Constraints on Supermassive Black Hole Binaries from the Gravitational-wave Background

El conjunto de datos de 15 años de NANOGrav muestra evidencias de la presencia de un fondo de ondas gravitacionales (GWB) de baja frecuencia. Aunque muchos procesos físicos pueden originar estas ondas gravitacionales de baja frecuencia, aquí analizamos la señal como procedente de una población de agujeros negros binarios supermasivos (SMBH) distribuidos por todo el Universo. Demostramos que los modelos astrofísicos de poblaciones binarias SMBH son capaces de reproducir tanto la amplitud como la forma del espectro de ondas gravitacionales de baja frecuencia observado. Aunque múltiples variaciones del modelo son capaces de reproducir el espectro GWB con nuestra precisión de medida actual, nuestros resultados subrayan la importancia de modelar con precisión la evolución binaria para producir espectros GWB realistas. Además, aunque unos parámetros razonables son capaces de reproducir las observaciones de 15 años, la amplitud implícita del GWB requiere que un gran número de parámetros se sitúen en los límites de los valores esperados o que un pequeño número de parámetros difieran notablemente de las expectativas estándar. Aunque todavía no somos capaces de establecer definitivamente el origen de la señal GWB inferida, la consistencia de la señal con las expectativas astrofísicas ofrece una perspectiva tentadora para confirmar que las binarias SMBH son capaces de formarse, alcanzar separaciones de sub-segundos y finalmente unirse. A medida que la importancia aumente con el tiempo, las características de orden superior del espectro del GWB determinarán definitivamente la naturaleza del GWB y permitirán nuevas restricciones sobre las poblaciones de SMBH. © 2023The NANOGrav 15 yr data set shows evidence for the presence of a low-frequency gravitational-wave background (GWB). While many physical processes can source such low-frequency gravitational waves, here we analyze the signal as coming from a population of supermassive black hole (SMBH) binaries distributed throughout the Universe. We show that astrophysically motivated models of SMBH binary populations are able to reproduce both the amplitude and shape of the observed low-frequency gravitational-wave spectrum. While multiple model variations are able to reproduce the GWB spectrum at our current measurement precision, our results highlight the importance of accurately modeling binary evolution for producing realistic GWB spectra. Additionally, while reasonable parameters are able to reproduce the 15 yr observations, the implied GWB amplitude necessitates either a large number of parameters to be at the edges of expected values or a small number of parameters to be notably different from standard expectations. While we are not yet able to definitively establish the origin of the inferred GWB signal, the consistency of the signal with astrophysical expectations offers a tantalizing prospect for confirming that SMBH binaries are able to form, reach subparsec separations, and eventually coalesce. As the significance grows over time, higher-order features of the GWB spectrum will definitively determine the nature of the GWB and allow for novel constraints on SMBH populations. © 2023. The Author(s). Published by the American Astronomical Society

Regulation of anti-apoptotic signaling by Kruppel-like factors 4 and 5 mediates lapatinib resistance in breast cancer

The Kruppel-like transcription factors (KLFs) 4 and 5 (KLF4/5) are coexpressed in mouse embryonic stem cells, where they function redundantly to maintain pluripotency. In mammary carcinoma, KLF4/5 can each impact the malignant phenotype, but potential linkages to drug resistance remain unclear. In primary human breast cancers, we observed a positive correlation between KLF4/5 transcript abundance, particularly in the human epidermal growth factor receptor 2 (HER2)-enriched subtype. Furthermore, KLF4/5 protein was rapidly upregulated in human breast cancer cells following treatment with the HER2/epidermal growth factor receptor inhibitor, lapatinib. In addition, we observed a positive correlation between these factors in the primary tumors of genetically engineered mouse models (GEMMs). In particular, the levels of both factors were enriched in the basal-like tumors of the C3(1) TAg (SV40 large T antigen transgenic mice under control of the C3(1)/prostatein promoter) GEMM. Using tumor cells derived from this model as well as human breast cancer cells, suppression of KLF4 and/or KLF5 sensitized HER2-overexpressing cells to lapatinib. Indicating cooperativity, greater effects were observed when both genes were depleted. KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL). Moreover, MCL1 was upregulated by lapatinib in a KLF4/5-dependent manner, and enforced expression of MCL1 in KLF4/5-deficient cells restored drug resistance. In addition, combined suppression of KLF4/5 in cultured tumor cells additively inhibited anchorage-independent growth, resistance to anoikis and tumor formation in immunocompromised mice. Consistent with their cooperative role in drug resistance and other malignant properties, KLF4/5 levels selectively stratified human HER2-enriched breast cancer by distant metastasis-free survival. These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies

First Discovery of a Fast Radio Burst at 350 MHz by the GBNCC Survey

We report the first discovery of a fast radio burst (FRB), FRB 20200125A, by the Green Bank Northern Celestial Cap (GBNCC) Pulsar Survey conducted with the Green Bank Telescope at 350 MHz. FRB 20200125A was detected at a Galactic latitude of 58.43 degrees with a dispersion measure of 179 pc cm$^{-3}$, while electron density models predict a maximum Galactic contribution of 25 pc cm$^{-3}$ along this line of sight. Moreover, no apparent Galactic foreground sources of ionized gas that could account for the excess DM are visible in multi-wavelength surveys of this region. This argues that the source is extragalactic. The maximum redshift for the host galaxy is $z_{max}=0.17$, corresponding to a maximum comoving distance of approximately 750 Mpc. The measured peak flux density for FRB 20200125A is 0.37 Jy, and we measure a pulse width of 3.7 ms, consistent with the distribution of FRB widths observed at higher frequencies. Based on this detection and assuming an Euclidean flux density distribution of FRBs, we calculate an all-sky rate at 350 MHz of $3.4^{+15.4}_{-3.3} \times 10^3$ FRBs sky$^{-1}$ day$^{-1}$ above a peak flux density of 0.42 Jy for an unscattered pulse having an intrinsic width of 5 ms, consistent with rates reported at higher frequencies. Given the recent improvements in our single-pulse search pipeline, we also revisit the GBNCC survey sensitivity to various burst properties. Finally, we find no evidence of interstellar scattering in FRB 20200125A, adding to the growing evidence that some FRBs have circumburst environments where free-free absorption and scattering are not significant.Comment: 15 pages, 6 figures, Submitted to Ap

The Green Bank North Celestial Cap Survey. IX. Timing Follow-up for 128 Pulsars

The Green Bank North Celestial Cap survey is one of the largest and most sensitive searches for pulsars and transient radio objects. Observations for the survey have finished; priorities have shifted toward long-term monitoring of its discoveries. In this study, we have developed a pipeline to handle large data sets of archival observations and connect them to recent, high-cadence observations taken using the Canadian Hydrogen Intensity Mapping Experiment telescope. This pipeline handles data for 128 pulsars and has produced measurements of spin, positional, and orbital parameters that connect data over observation gaps as large as 2000 days. We have also measured glitches in the timing residuals for five of the pulsars included and proper motion for 19 sources (13 new). We include updates to orbital parameters for 19 pulsars, including nine previously unpublished binaries. For two of these binaries, we provide updated measurements of post-Keplerian binary parameters, which result in much more precise estimates of the total masses of both systems. For PSR J0509+3801, the much improved measurement of the Einstein delay yields much improved mass measurements for the pulsar and its companion, 1.399(6) M⊙ and 1.412(6) M⊙, respectively. For this system, we have also obtained a measurement of the orbital decay due to the emission of gravitational waves

Phosphoproteomics-Based Modeling Defines the Regulatory Mechanism Underlying Aberrant EGFR Signaling

BACKGROUND: Mutation of the epidermal growth factor receptor (EGFR) results in a discordant cell signaling, leading to the development of various diseases. However, the mechanism underlying the alteration of downstream signaling due to such mutation has not yet been completely understood at the system level. Here, we report a phosphoproteomics-based methodology for characterizing the regulatory mechanism underlying aberrant EGFR signaling using computational network modeling. METHODOLOGY/PRINCIPAL FINDINGS: Our phosphoproteomic analysis of the mutation at tyrosine 992 (Y992), one of the multifunctional docking sites of EGFR, revealed network-wide effects of the mutation on EGF signaling in a time-resolved manner. Computational modeling based on the temporal activation profiles enabled us to not only rediscover already-known protein interactions with Y992 and internalization property of mutated EGFR but also further gain model-driven insights into the effect of cellular content and the regulation of EGFR degradation. Our kinetic model also suggested critical reactions facilitating the reconstruction of the diverse effects of the mutation on phosphoproteome dynamics. CONCLUSIONS/SIGNIFICANCE: Our integrative approach provided a mechanistic description of the disorders of mutated EGFR signaling networks, which could facilitate the development of a systematic strategy toward controlling disease-related cell signaling

The NANOGrav 15 yr Data Set: Search for Transverse Polarization Modes in the Gravitational-wave Background

\ua9 2024. The Author(s). Published by the American Astronomical Society.Recently we found compelling evidence for a gravitational-wave background with Hellings and Downs (HD) correlations in our 15 yr data set. These correlations describe gravitational waves as predicted by general relativity, which has two transverse polarization modes. However, more general metric theories of gravity can have additional polarization modes, which produce different interpulsar correlations. In this work, we search the NANOGrav 15 yr data set for evidence of a gravitational-wave background with quadrupolar HD and scalar-transverse (ST) correlations. We find that HD correlations are the best fit to the data and no significant evidence in favor of ST correlations. While Bayes factors show strong evidence for a correlated signal, the data does not strongly prefer either correlation signature, with Bayes factors ∼2 when comparing HD to ST correlations, and ∼1 for HD plus ST correlations to HD correlations alone. However, when modeled alongside HD correlations, the amplitude and spectral index posteriors for ST correlations are uninformative, with the HD process accounting for the vast majority of the total signal. Using the optimal statistic, a frequentist technique that focuses on the pulsar-pair cross-correlations, we find median signal-to-noise ratios of 5.0 for HD and 4.6 for ST correlations when fit for separately, and median signal-to-noise ratios of 3.5 for HD and 3.0 for ST correlations when fit for simultaneously. While the signal-to-noise ratios for each of the correlations are comparable, the estimated amplitude and spectral index for HD are a significantly better fit to the total signal, in agreement with our Bayesian analysis

FGFR3, HRAS, KRAS, NRAS and PIK3CA Mutations in Bladder Cancer and Their Potential as Biomarkers for Surveillance and Therapy

Background: Fifty percent of patients with muscle-invasive bladder cancer (MI-BC) die from their disease and current chemotherapy treatment only marginally increases survival. Novel therapies targeting receptor tyrosine kinases or activated oncogenes may improve outcome. Hence, it is necessary to stratify patients based on mutations in relevant oncogenes. Patients with non-muscle-invasive bladder cancer (NMI-BC) have excellent survival, however two-thirds develop recurrences. Tumor specific mutations can be used to detect recurrences in urine assays, presenting a more patient-friendly diagnostic procedure than cystoscopy. Methodology/Principal Findings: To address these issues, we developed a mutation assay for the simultaneous detection of 19 possible mutations in the HRAS, KRAS, and NRAS genes. With this assay and mutation assays for the FGFR3 and PIK3CA oncogenes, we screened primary bladder tumors of 257 patients and 184 recurrences from 54 patients. Additionally, in primary tumors p53 expression was obtained by immunohistochemistry. Of primary tumors 64% were mutant for FGFR3, 11% for RAS, 24% for PIK3CA, and 26% for p53. FGFR3 mutations were mutually exclusive with RAS mutations (p = 0.001) and co-occurred with PIK3CA mutations (p = 0.016). P53 overexpression was mutually exclusive with PIK3CA and FGFR3 mutations (p≤0.029). Mutations in the RAS and PIK3CA genes were not predictors for recurrence-free, progression-free and disease-specific survival. In patients presenting with NMI-BC grade 3 and MI-BC, 33 and 36% of the primary tumors were mutant. In patients with low-grade NMI-BC, 88% of the primary tumors carried a mutation and 88% of the recurrences were mutant. Conclusions/Significance: The mutation assays present a companion diagnostic to define patients for targeted therapies. In addition, the assays are a potential biomarker to detect recurrences during surveillance. We showed that 88% of patients presenting with low-grade NMI-BC are eligible for such a follow-up. This may contribute to a reduction in the number of cystoscopical examinations