Direct knockdown of phospho-PTM targets mediated by TRIM21 can improve personalized treatment in breast cancer.

PURPOSE: In this work for the first time, we showed specific and direct knockdown of important oncogenic proteins of interest and their phospho-PTM targets in tripartite motif containing-21 (TRIM21) overexpressing breast cancer (BC) cells. We revealed the functional and therapeutic consequences of this protein knockdown approach called 'TRIM-ing'. METHODS: To target HER2, HER3, STAT3 or their activated forms, electroporation and puls-in transfection were standardized for mAb delivery in AU565 and MCF7 BC cell lines. Cancer cells were treated with HER2-targeted medicines (Trastuzumab and Neratinib) or STAT3 targeted inhibitors (Stattic and Niclosamide) with or without respective target TRIM-ing. Real-time PCR, immunoblotting, immunofluorescence, cytotoxicity, short- and long-term cell survival assessments were done following standard methodologies. 3-D structure modelling was used to verify the binding of mAb onto the STAT3 target. RESULTS: TRIM-ing of HER2 or HER3 receptors or their activated phospho-forms in BC cells showed rapid degradation of respective protein forms, shattering down the downstream signaling (p-ERK, p-AKT) that lasts for up to 7-8 days. This significantly inhibited BC survival (p < 0.001), showing a synergistic therapeutic effect with HER2 medicine trastuzumab or neratinib. Additionally, specific TRIM-ing ability of canonical pY705 or non-canonical pS727 PTMs of STAT3 protein was demonstrated in MCF7 cells, causing significant cytotoxicity (p < 0.05). TRIM-ing of STAT3 PTM, when combined with the same PTM-specific inhibitors, a synergistic treatment effect was observed. CONCLUSION: The work demonstrated that TRIM-ing could directly reduce various oncogenic targets or their specific activated form inside the cancer cells without compensatory pathway activation, a conundrum limiting the therapeutic benefit of current personalized medicines.

Pluto near the edge of chaos.

Many of the unusual properties of Pluto's orbit are widely accepted as evidence for the orbital migration of the giant planets in early solar system history. However, some properties remain an enigma. Pluto's long-term orbital stability is supported by two special properties of its orbit that limit the location of its perihelion in azimuth and in latitude. We revisit Pluto's orbital dynamics with a view to elucidating the individual and collective gravitational effects of the giant planets on constraining its perihelion location. While the resonant perturbations from Neptune account for the azimuthal constraint on Pluto's perihelion location, we demonstrate that the long-term and steady persistence of the latitudinal constraint is possible only in a narrow range of additional secular forcing which arises fortuitously from the particular orbital architecture of the other giant planets. Our investigations also find that Jupiter has a largely stabilizing influence whereas Uranus has a largely destabilizing influence on Pluto's orbit. Overall, Pluto's orbit is rather surprisingly close to a zone of strong chaos.

New results on orbital resonances.

Perturbative analyses of planetary resonances commonly predict singularities and/or divergences of resonance widths at very low and very high eccentricities. We have recently reexamined the nature of these divergences using non-perturbative numerical analyses, making use of Poincaré sections but from a different perspective relative to previous implementations of this method. This perspective reveals fine structure of resonances which otherwise remains hidden in conventional approaches, including analytical, semi-analytical and numerical-averaging approaches based on the critical resonant angle. At low eccentricity, first order resonances do not have diverging widths but have two asymmetric branches leading away from the nominal resonance location. A sequence of structures called "low-eccentricity resonant bridges" connecting neighboring resonances is revealed. At planet-grazing eccentricity, the true resonance width is non-divergent. At higher eccentricities, the new results reveal hitherto unknown resonant structures and show that these parameter regions have a loss of some - though not necessarily entire - resonance libration zones to chaos. The chaos at high eccentricities was previously attributed to the overlap of neighboring resonances. The new results reveal the additional role of bifurcations and co-existence of phase-shifted resonance zones at higher eccentricities. By employing a geometric point of view, we relate the high eccentricity phase space structures and their transitions to the shapes of resonant orbits in the rotating frame. We outline some directions for future research to advance understanding of the dynamics of mean motion resonances.

Dynamical instabilities in systems of multiple short-period planets are likely driven by secular chaos: a case study of Kepler-102.

We investigated the dynamical stability of high-multiplicity Kepler and K2 planetary systems. Our numerical simulations find instabilities in ~ 20% of the cases on a wide range of timescales (up to 5×109 orbits) and over an unexpectedly wide range of initial dynamical spacings. To identify the triggers of long-term instability in multi-planet systems, we investigated in detail the five-planet Kepler-102 system. Despite having several near-resonant period ratios, we find that mean motion resonances are unlikely to directly cause instability for plausible planet masses in this system. Instead, we find strong evidence that slow inward transfer of angular momentum deficit (AMD) via secular chaos excites the eccentricity of the innermost planet, Kepler-102 b, eventually leading to planet-planet collisions in ~ 80% of Kepler-102 simulations. Kepler-102 b likely needs a mass ≳ 0.1M ⊕, hence a bulk density exceeding about half Earth's, in order to avoid dynamical instability. To investigate the role of secular chaos in our wider set of simulations, we characterize each planetary system's AMD evolution with a "spectral fraction" calculated from the power spectrum of short integrations (~ 5 × 106 orbits). We find that small spectral fractions (≲ 0.01) are strongly associated with dynamical stability on long timescales (5 × 109 orbits) and that the median time to instability decreases with increasing spectral fraction. Our results support the hypothesis that secular chaos is the driver of instabilities in many non-resonant multi-planet systems, and also demonstrate that the spectral analysis method is an efficient numerical tool to diagnose long term (in)stability of multi-planet systems from short simulations.

Noncanonical pS727 post translational modification dictates major STAT3 activation and downstream functions in breast cancer.

Activation of STAT3 via Y705-phosphorylation is well documented across multiple cancer types and thus forms the basis of canonical pathway to judge STAT3 activation. Recently, important roles of two other post translational modification (PTM) sites, i.e. S727-phosphorylation and K685-acetylation, leading to STAT3 activation are reported. However, their critical mode of function in controlling STAT3 dimerization and signaling, independent of canonical activation remains elusive. Therefore, to understand the functional relevance of each STAT3 PTMs in breast cancer (BC), cell models are developed by stable overexpression of PTM-site specific point mutants, i.e. Y705F, S727A or K685R, in a 3'UTR-STAT3 knockdown BC cell background. Results using this model system reveal novel findings showing that phosphorylation at S727 can lead to STAT3 activation independent of phosphoY705. We also demonstrate that loss of pS727 or K685ac significantly affects functional phenotypes such as cell survival and proliferation as well as downstream transcriptional activity (Twist 1, Socs3, c-Myc, Bcl-1 and Mcl-1) of STAT3. Thereafter, by utilizing a BRET biosensor for measuring STAT3 phosphorylation in live cells, a crucial role of pS727 in dictating STAT3 activation and homodimerization formation is uncovered. Further by performing retrospective IHC analysis of total and phospho-forms of STAT3 in a cohort of 76 triple negative breast cancer (TNBC) patient samples, a significant dominant expression of phosphoS727 over phosphoY705 PTM (p < 0.001) is found in STAT3 positive cases. We also focus on validating known STAT3 inhibitor molecules for their action against both pY705 and pS727 activation. This study for the first time demonstrates that an anti-helminth drug compound, Niclosamide, is capable of inactivating both phospho-PTM sites on STAT3 and exhibits excellent anticancer efficacy in preclinical TNBC tumour model.

Not a simple relationship between Neptune's migration speed and Kuiper belt inclination excitation.

We present numerical simulations of giant planet migration in our solar system and examine how the speed of planetary migration affects inclinations in the resulting population of small bodies (test particles) scattered outward and subsequently captured into Neptune's 3:2 mean motion resonance (the Plutinos) as well as the hot classical Kuiper belt population. We do not find a consistent relationship between the degree of test particle inclination excitation and e-folding planet migration timescales in the range 5 - 50 Myr. Our results present a counter-example to Nesvorný (2015)'s finding that the Plutino and hot classical inclinations showed a marked increase with increasing e-folding timescales for Neptune's migration. We argue that these differing results are likely due to differing secular architectures of the giant planets during and after migration. Small changes in the planets' initial conditions and differences in the numerical implementation of planet migration can result in different amplitudes of the planets' inclination secular modes, and this can lead to different final inclination distributions for test particles in the simulations. We conclude that the observed large inclination dispersion of Kuiper belt objects does not require Neptune's migration to be slow; planetary migration with e-folding timescales of 5, 10, 30, and 50 Myr can all yield inclination dispersions similar to the observed Plutino and hot classical populations, with no correlation between the degree of inclination excitation and migration speed.

Resonant Kuiper belt objects: a review.

Our understanding of the history of the solar system has undergone a revolution in recent years, owing to new theoretical insights into the origin of Pluto and the discovery of the Kuiper belt and its rich dynamical structure. The emerging picture of dramatic orbital migration of the planets driven by interaction with the primordial Kuiper belt is thought to have produced the final solar system architecture that we live in today. This paper gives a brief summary of this new view of our solar system's history and reviews the astronomical evidence in the resonant populations of the Kuiper belt.

Nuclear matrix-associated protein SMAR1 regulates alternative splicing via HDAC6-mediated deacetylation of Sam68.

Pre-mRNA splicing is a complex regulatory nexus modulated by various trans-factors and their posttranslational modifications to create a dynamic transcriptome through alternative splicing. Signal-induced phosphorylation and dephosphorylation of trans-factors are known to regulate alternative splicing. However, the role of other posttranslational modifications, such as deacetylation/acetylation, methylation, and ubiquitination, that could modulate alternative splicing in either a signal-dependent or -independent manner remain enigmatic. Here, we demonstrate that Scaffold/matrix-associated region-binding protein 1 (SMAR1) negatively regulates alternative splicing through histone deacetylase 6 (HDAC6)-mediated deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa). SMAR1 is enriched in nuclear splicing speckles and associates with the snRNAs that are involved in splice site recognition. ERK-MAPK pathway that regulates alternative splicing facilitates ERK-1/2-mediated phosphorylation of SMAR1 at threonines 345 and 360 and localizes SMAR1 to the cytoplasm, preventing its interaction with Sam68. We showed that endogenously, SMAR1 through HDAC6 maintains Sam68 in a deacetylated state. However, knockdown or ERK-mediated phosphorylation of SMAR1 releases the inhibitory SMAR1-HDAC6-Sam68 complex, facilitating Sam68 acetylation and alternative splicing. Furthermore, loss of heterozygosity at the Chr.16q24.3 locus in breast cancer cells, wherein the human homolog of SMAR1 (BANP) has been mapped, enhances Sam68 acetylation and CD44 variant exon inclusion. In addition, tail-vein injections in mice with human breast cancer MCF-7 cells depleted for SMAR1 showed increased CD44 variant exon inclusion and concomitant metastatic propensity, confirming the functional role of SMAR1 in regulation of alternative splicing. Thus, our results reveal the complex molecular mechanism underlying SMAR1-mediated signal-dependent and -independent regulation of alternative splicing via Sam68 deacetylation.

In vivo analysis of biodegradable liposome gold nanoparticles as efficient agents for photothermal therapy of cancer.

We report biodegradable plasmon resonant liposome gold nanoparticles (LiposAu NPs) capable of killing cancer cells through photothermal therapy. The pharmacokinetic study of LiposAu NPs performed in a small animal model indicates in situ degradation in hepatocytes and further getting cleared through the hepato-biliary and renal route. Further, the therapeutic potential of LiposAu NPs tested in mouse tumor xenograft model using NIR laser (750 nm) illumination resulted in complete ablation of the tumor mass, thus prolonging disease-free survival.

IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples.

In anatomic pathology, immunohistochemistry (IHC) serves as a diagnostic and prognostic method for identification of disease markers in tissue samples that directly influences classification and grading the disease, influencing patient management. However, till today over most of the world, pathological analysis of tissue samples remained a time-consuming and subjective procedure, wherein the intensity of antibody staining is manually judged and thus scoring decision is directly influenced by visual bias. This instigated us to design a simple method of automated digital IHC image analysis algorithm for an unbiased, quantitative assessment of antibody staining intensity in tissue sections. As a first step, we adopted the spectral deconvolution method of DAB/hematoxylin color spectra by using optimized optical density vectors of the color deconvolution plugin for proper separation of the DAB color spectra. Then the DAB stained image is displayed in a new window wherein it undergoes pixel-by-pixel analysis, and displays the full profile along with its scoring decision. Based on the mathematical formula conceptualized, the algorithm is thoroughly tested by analyzing scores assigned to thousands (n = 1703) of DAB stained IHC images including sample images taken from human protein atlas web resource. The IHC Profiler plugin developed is compatible with the open resource digital image analysis software, ImageJ, which creates a pixel-by-pixel analysis profile of a digital IHC image and further assigns a score in a four tier system. A comparison study between manual pathological analysis and IHC Profiler resolved in a match of 88.6% (P<0.0001, CI = 95%). This new tool developed for clinical histopathological sample analysis can be adopted globally for scoring most protein targets where the marker protein expression is of cytoplasmic and/or nuclear type. We foresee that this method will minimize the problem of inter-observer variations across labs and further help in worldwide patient stratification potentially benefitting various multinational clinical trial initiatives.

Quantitative immunohistochemical analysis reveals association between sodium iodide symporter and estrogen receptor expression in breast cancer.

BACKGROUND: Human sodium iodide symporter (hNIS) gene over-expression is under active consideration worldwide as an alternative target molecule for breast cancer (BC) diagnosis and targeted radio-iodine treatment. However, the field demands better stratified analysis of endogenous hNIS expression across major BC subtypes. Therefore, we have analyzed subtype-specific variation of hNIS overexpression in breast tumor tissue samples by immunohistochemistry (IHC) and also report the development of a homogeneous, quantitative analysis method of digital IHC images. METHODS: hNIS expression was analyzed from 108 BC tissue samples by IHC. Sub-cellular localization of hNIS protein was analyzed by dual immunofluorescence (IF) staining method using hNIS and HER2 antibodies. An ImageJ based two-step digital analysis method was developed and applied for the bias-free analysis of the images. RESULTS: Staining of the tumor samples show 70% cases are hNIS positive indicating high incidence of hNIS positive cases in BC. More importantly, a subtype specific analysis done for the first time shows that hNIS expression is overly dominated in estrogen receptor (ER) positive cases than the receptor negative cases. Further, 56% of the ER+ve, PgR+ve, HER2-ve and 36% of ER+ve, PgR+ve, HER2+ve cases show highest intensity staining equivalent to the thyroid tissue. A significant positive correlation is also observed between hNIS and estrogen receptor expression (p = 0.0033, CI = 95%) suggesting hNIS mediated targeted radio-iodine therapy procedures may benefit both ER+ve, PgR+ve, HER2-ve as well as HER2+ve cases. Further, in a few cases, hNIS and HER2 protein localization is demonstrated by overlapping membrane co-expression. ImageJ based image analysis method shows over 70% match with manual pathological scoring method. CONCLUSION: The study indicates a positive link between hNIS and ER expression in BC. The quantitative IHC image analysis method reported here will further help in patient stratification and potentially benefit global clinical assessment where hNIS mediated targeted ¹³¹I radio-ablative therapy is aimed.

Chaotic exchange of solid material between planetary systems: implications for lithopanspermia.

We examined a low-energy mechanism for the transfer of meteoroids between two planetary systems embedded in a star cluster using quasi-parabolic orbits of minimal energy. Using Monte Carlo simulations, we found that the exchange of meteoroids could have been significantly more efficient than previously estimated. Our study is relevant to astrobiology, as it addresses whether life on Earth could have been transferred to other planetary systems in the Solar System's birth cluster and whether life on Earth could have been transferred from beyond the Solar System. In the Solar System, the timescale over which solid material was delivered to the region from where it could be transferred via this mechanism likely extended to several hundred million years (as indicated by the 3.8-4.0 Ga epoch of the Late Heavy Bombardment). This timescale could have overlapped with the lifetime of the Solar birth cluster (∼100-500 Myr). Therefore, we conclude that lithopanspermia is an open possibility if life had an early start. Adopting parameters from the minimum mass solar nebula, considering a range of planetesimal size distributions derived from observations of asteroids and Kuiper Belt objects and theoretical coagulation models, and taking into account Oort Cloud formation models, we discerned that the expected number of bodies with mass>10 kg that could have been transferred between the Sun and its nearest cluster neighbor could be of the order of 10(14) to 3·10(16), with transfer timescales of tens of millions of years. We estimate that of the order of 3·10(8)·l (km) could potentially be life-bearing, where l is the depth of Earth's crust in kilometers that was ejected as the result of the early bombardment.

Long-term cycling of Kozai-Lidov cycles: extreme eccentricities and inclinations excited by a distant eccentric perturber.

The very long-term evolution of the hierarchical restricted three-body problem is calculated analytically for high inclinations. The Kozai-Lidov Cycles (KLCs) slowly evolve due to the octupole term in the perturber's potential and exhibit striking features, including extremely high eccentricities and the generation of retrograde orbits with respect to the perturber. These features were found in recent numerical experiments of the nonrestricted three-body problem and were attributed inaccurately to the comparable and low masses of the two orbiting companions. Our calculation is done by averaging for the first time the double averaged secular equations of motion over the KLCs and finding a new constant of the motion. These very long-term effects are likely to be important in various astrophysical systems thought to involve KLCs, such as hot Jupiters, irregular moons of planets, and many others.

A record of planet migration in the main asteroid belt.

The main asteroid belt lies between the orbits of Mars and Jupiter, but the region is not uniformly filled with asteroids. There are gaps, known as the Kirkwood gaps, in distinct locations that are associated with orbital resonances with the giant planets; asteroids placed in these locations will follow chaotic orbits and be removed. Here we show that the observed distribution of main belt asteroids does not fill uniformly even those regions that are dynamically stable over the age of the Solar System. We find a pattern of excess depletion of asteroids, particularly just outward of the Kirkwood gaps associated with the 5:2, the 7:3 and the 2:1 Jovian resonances. These features are not accounted for by planetary perturbations in the current structure of the Solar System, but are consistent with dynamical ejection of asteroids by the sweeping of gravitational resonances during the migration of Jupiter and Saturn approximately 4 Gyr ago.

The origin of planetary impactors in the inner solar system.

Insights into the history of the inner solar system can be derived from the impact cratering record of the Moon, Mars, Venus, and Mercury and from the size distributions of asteroid populations. Old craters from a unique period of heavy bombardment that ended approximately 3.8 billion years ago were made by asteroids that were dynamically ejected from the main asteroid belt, possibly due to the orbital migration of the giant planets. The impactors of the past approximately 3.8 billion years have a size distribution quite different from that of the main belt asteroids but very similar to that of near-Earth asteroids.