The Role of Spy1 in Mammary Morphogenesis

Cell growth and development is regulated by the cell division cycle, which dictates how efficiently cells communicate with each other and is dependent on cellular morphology. Our lab focuses on a novel cell cycle regulator, Spy1A, which has been shown to enhance cellular proliferation and regulate mammary development. Elevated levels of Spy1A significantly increased cell invasion, coincident with an increase beta-catenin transcriptional levels. We hypothesize that these characteristics may maintain cells in a more & stem-like state. Mammary stem cells are highly proliferative, they rely on adherence to surrounding cells for self renewal and invasion and migration into the mammary fat pad. We further demonstrate that Spy1A overexpression results in the formation of larger mammospheres, where mammosphere size has been correlative of stimulating stem cell self-renewal. Further resolving the roles of Spy1 in the developing mammary gland is essential to fully elucidate its roles in human disease

The cyclin-like protein Spy1/RINGO promotes mammary transformation and is elevated in human breast cancer

Abstract Background Spy1 is a novel \u27cyclin-like\u27 activator of the G1/S transition capable of enhancing cell proliferation as well as inhibiting apoptosis. Spy1 protein levels are tightly regulated during normal mammary development and forced overexpression in mammary mouse models accelerates mammary tumorigenesis. Methods Using human tissue samples, cell culture models and in vivo analysis we study the implications of Spy1 as a mediator of mammary transformation and breast cancer proliferation. Results We demonstrate that this protein can facilitate transformation in a manner dependent upon the activation of the G2/M Cdk, Cdk1, and the subsequent inhibition of the anti-apoptotic regulator FOXO1. Importantly, we show for the first time that enhanced levels of Spy1 protein are found in a large number of human breast cancers and that knockdown of Spy1 impairs breast cancer cell proliferation. Conclusions Collectively, this work supports that Spy1 is a unique activator of Cdk1 in breast cancer cells and may represent a valuable drug target and/or a prognostic marker for subsets of breast cancers

The Mechanisms of Zinc Action as a Potent Anti-Viral Agent: The Clinical Therapeutic Implication in COVID-19

The pandemic of COVID-19 was caused by a novel coronavirus termed as SARS-CoV2 and is still ongoing with high morbidity and mortality rates in the whole world. The pathogenesis of COVID-19 is highly linked with over-active immune and inflammatory responses, leading to activated cytokine storm, which contribute to ARDS with worsen outcome. Currently, there is no effective therapeutic drug for the treatment of COVID-19. Zinc is known to act as an immune modulator, which plays an important role in immune defense system. Recently, zinc has been widely considered as an anti-inflammatory and anti-oxidant agent. Accumulating numbers of studies have revealed that zinc plays an important role in antiviral immunity in several viral infections. Several early clinical trials clearly indicate that zinc treatment remarkably decreased the severity of the upper respiratory infection of rhinovirus in humans. Currently, zinc has been used for the therapeutic intervention of COVID-19 in many different clinical trials. Several clinical studies reveal that zinc treatment using a combination of HCQ and zinc pronouncedly reduced symptom score and the rates of hospital admission and mortality in COVID-19 patients. These data support that zinc might act as an anti-viral agent in the addition to its anti-inflammatory and anti-oxidant properties for the adjuvant therapeutic intervention of COVID-19

A kinome screen identifies checkpoint kinase 1 (CHK1) as a sensitizer for RRM1-dependent gemcitabine efficacy.

Gemcitabine is among the most efficacious and widely used antimetabolite agents. Its molecular targets are ribonucleotide reductase M1 (RRM1) and elongating DNA. Acquired and de novo resistance as a result of RRM1 overexpression are major obstacles to therapeutic efficacy. We deployed a synthetic lethality screen to investigate if knockdown of 87 selected protein kinases by siRNA could overcome RRM1-dependent gemcitabine resistance in high and low RRM1-expressing model systems. The models included genetically RRM1-modified lung and breast cancer cell lines, cell lines with gemcitabine-induced RRM1 overexpression, and a series of naturally gemcitabine-resistant cell lines. Lead molecular targets were validated by determination of differential gemcitabine activity using cell lines with and without target knock down, and by assessing synergistic activity between gemcitabine and an inhibitor of the lead target. CHK1 was identified has the kinase with the most significant and robust interaction, and it was validated using AZD7762, a small-molecule ATP-competitive inhibitor of CHK1 activation. Synergism between CHK1 inhibition and RRM1-dependent gemcitabine efficacy was observed in cells with high RRM1 levels, while antagonism was observed in cells with low RRM1 levels. In addition, four cell lines with natural gemcitabine resistance demonstrated improved gemcitabine efficacy after CHK1 inhibition. In tumor specimens from 187 patients with non-small-cell lung cancer, total CHK1 and RRM1 in situ protein levels were significantly (p = 0.003) and inversely correlated. We conclude that inhibition of CHK1 may have its greatest clinical utility in malignancies where gemcitabine resistance is a result of elevated RRM1 levels. We also conclude that CHK1 inhibition in tumors with low RRM1 levels may be detrimental to gemcitabine efficacy

The CHK1 inhibitor AZD7762 increases gemcitabine efficacy.

<p>Four gemcitabine-resistant clones were exposed to drugs for 4 days. Data points are the mean of four replicates, and bars depict the standard error. The IC50 values were as follows: For clone H23-G-C8, control 51.4 nM, AZD7762-150 nM 22.8 nM, AZD7762-300 nM 8.5 nM; for clone H23-G-C23, control 47.7 nM, AZD7762-150 nM 22.6 nM, AZD7762-300 nM 13.2 nM; for clone H1299-G-C2, control 186.1 nM, AZD7762-150 nM 104.8 nM, AZD7762-300 nM 90.1 nM; for clone H1299-G-C18, control 123.8 nM, AZD7762-150 nM 52.0 nM, AZD7762-300 nM 42.0 nM.</p

Gemcitabine and CHK1 inhibitor (AZD7762) combination index values in gemcitabine-resistant cell lines and clones.

<p>Gemcitabine and CHK1 inhibitor (AZD7762) combination index values in gemcitabine-resistant cell lines and clones.</p

CHK1 and TEC knockdown and RRM1-dependent gemcitabine sensitivity.

<p>The H23 and MCF7 cell line systems with increased (−R1) and reduced (−shR1) RRM1 expression were transfected with CHK1-specific or TEC-specific siRNA and exposed to gemcitabine. IC50 values are the mean of four replicates. CHK1 is depicted in green and red, TEC is depicted in blue and orange.</p

Interaction of selected kinases with RRM1-dependent gemcitabine efficacy.

*<p>The IC50 ratio was calculated by dividing the siRNA-specific IC50 by the control IC50. Cell lines H23, MCF7, and H1299 were exposed to gemcitabine for four 4 days. H125, H650, H1648, and H2122 were exposed for 5 to 7 days (a 4-day exposure yields IC50 values above 1 mM). RRM1 knockdown in the gemcitabine resistant clones restored sensitivity (IC50 ratios: 0.15 for H23-G-C8 and H23-G-C23, 0.19 for H1299-G-C2 and 0.09 for H1299-G-C18).</p

Synthetic lethality of 87 kinases as a function of RRM1 expression.

<p>Two cell line systems, H23 (blue bars) and MCF7 (red bars), with stably increased or reduced RRM1 expression were transfected with gene-specific siRNAs and viability was scored. Bars to the left indicate greater lethality in high compared to low RRM1-expressing cells. Bars to the right indicate greater lethality in low compared to high RRM1-expressing cells. Bars below the x-axis indicate one (wide bar) or two (narrow bar) standard deviations in the lethality scores for the two cell line systems respectively.</p