Redox regulation of Rac1 by thiol oxidation

The Rac1 GTPase is an essential and ubiquitous protein that signals through numerous pathways to control critical cellular processes, including cell growth, morphology, and motility. Rac1 deletion is embryonic lethal, and its dysregulation or mutation can promote cancer, arthritis, cardiovascular disease, and neurological disorders. Rac1 activity is highly regulated by modulatory proteins and posttranslational modifications. Whereas much attention has been devoted to guanine nucleotide exchange factors that act on Rac1 to promote GTP loading and Rac1 activation, cellular oxidants may also regulate Rac1 activation by promoting guanine nucleotide exchange. Herein, we show that Rac1 contains a redox-sensitive cysteine (Cys18) that can be selectively oxidized at physiological pH because of its lowered pKa. Consistent with these observations, we show that Rac1 is glutathiolated in primary chondrocytes. Oxidation of Cys18 by glutathione greatly perturbs Rac1 guanine nucleotide binding and promotes nucleotide exchange. As aspartate substitutions have been previously used to mimic cysteine oxidation, we characterized the biochemical properties of Rac1C18D. We also evaluated Rac1C18S as a redox-insensitive variant and found that it retains structural and biochemical properties similar to those of Rac1WT but is resistant to thiol oxidation. In addition, Rac1C18D, but not Rac1C18S, shows greatly enhanced nucleotide exchange, similar to that observed for Rac1 oxidation by glutathione. We employed Rac1C18D in cell-based studies to assess whether this fast-cycling variant, which mimics Rac1 oxidation by glutathione, affects Rac1 activity and function. Expression of Rac1C18D in Swiss 3T3 cells showed greatly enhanced GTP-bound Rac1 relative to Rac1WT and the redox-insensitive Rac1C18S variant. Moreover, expression of Rac1C18D in HEK-293T cells greatly promoted lamellipodia formation. Our results suggest that Rac1 oxidation at Cys18 is a novel posttranslational modification that upregulates Rac1 activity

CDK4/6 inhibition triggers anti-tumor immunity

Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the initiation and progression of various malignancies1,2. Pharmacologic inhibitors of CDK4/6 have shown significant activity against several solid tumors3,4. Their primary mechanism of action is thought to be the inhibition of phosphorylation of the retinoblastoma (RB) tumor suppressor, inducing G1 cell cycle arrest in tumor cells5. Here, we use murine models of breast carcinoma and other solid tumors to show that selective CDK4/6 inhibitors not only induce tumor cell cycle arrest, but also promote anti-tumor immunity. We confirm this phenomenon through transcriptomic analysis of serial biopsies from a clinical trial of CDK4/6 inhibitor treatment for breast cancer. The enhanced anti-tumor immune response has two underpinnings. First, CDK4/6 inhibitors activate tumor cell expression of endogenous retroviral elements, thus increasing intracellular levels of double-stranded RNA. This in turn stimulates production of type III interferons and hence enhances tumor antigen presentation. Second, CDK4/6 inhibitors markedly suppress the proliferation of regulatory T cells (Tregs). Mechanistically, the effects of CDK4/6 inhibitors on both tumor cells and Tregs are associated with reduced activity of the E2F target, DNA methyltransferase 1. Ultimately, these events promote cytotoxic T cell-mediated clearance of tumor cells, which is further enhanced by the addition of immune checkpoint blockade. Our findings indicate that CDK4/6 inhibitors increase tumor immunogenicity and provide rationale for new combination regimens comprising CDK4/6 inhibitors and immunotherapies as anti-cancer treatment

Accounting for tumor heterogeneity when using CRISPR-Cas9 for cancer progression and drug sensitivity studies

Gene editing protocols often require the use of a subcloning step to isolate successfully edited cells, the behavior of which is then compared to the aggregate parental population and/or other non-edited subclones. Here we demonstrate that the inherent functional heterogeneity present in many cell lines can render these populations inappropriate controls, resulting in erroneous interpretations of experimental findings. We describe a novel CRISPR/Cas9 protocol that incorporates a single-cell cloning step prior to gene editing, allowing for the generation of appropriately matched, functionally equivalent control and edited cell lines. As a proof of concept, we generated matched control and osteopontin-knockout Her2+ and Estrogen receptor-negative murine mammary carcinoma cell lines and demonstrated that the osteopontin-knockout cell lines exhibit the expected biological phenotypes, including unaffected primary tumor growth kinetics and reduced metastatic outgrowth in female FVB mice. Using these matched cell lines, we discovered that osteopontin-knockout mammary tumors were more sensitive than control tumors to chemotherapy in vivo. Our results demonstrate that heterogeneity must be considered during experimental design when utilizing gene editing protocols and provide a solution to account for it

Osakeyhtiön tasekirjapohja: Case: Kouvola Innovation Oy

Kunnan Taitoa Oy:n Kouvolan toimipisteen asiakkaan, Kouvolan kaupungin omistaman yhtiön, Kouvola Innovation Oy:n kirjanpitoa on hoitanut useampi eri henkilö, eikä tilinpäätöstä ja tasekirjaa varten ole ollut valmista pohjaa tai yhtenäistä mallia. Eri vuosien tasekirjoissa on eroja sekä ulkonäössä että sisällössä. Tämän opinnäytetyön tavoitteena oli luoda Kunnan Taitoa Oy:n Kouvolan toimipisteeseen tasekirjapohja vanhoja tasekirjoja kehittämällä. Pohja tulisi Kouvola Innovation Oy:n tilinpäätöksen laatijan apuvälineeksi. Tarkoituksena oli tehdä pohjasta sellainen, että sitä voidaan pienillä muutoksilla hyödyntää muidenkin yhtiöiden tilinpäätökseen ja että se hakee tilinpäätöslaskelmien luvut suoraan konsernikirjanpito-ohjelman tietokannasta. Työssä perehdytään tilinpäätöksen ja tasekirjan sisältöön lainsäädännön, asetuksien, yleisohjeiden ja kirjallisuuden avulla. Teoriaa on kirjoitettu samaan aikaan, kun tasekirjapohjaa on laadittu. Aikaisempia tasekirjoja tutkimalla ja teoriaa selvittämällä pyrittiin löytämään kehittämiskohtia niin ulkoasusta kuin asiasisällöstä. Työn tuloksena on toimiva tasekirjapohja, jota voidaan hyödyntää pienillä muutoksilla muidenkin yhtiöiden, kuin Kouvola Innovation Oy:n tilinpäätökseen. Pohja saatiin rakennettua niin, että se poimii laskelmiin ja liitetietoihin lukuja tietokannasta. Kehittämiskohtia ja niiden ratkaisuja löydettiin sekä niihin liittyviä tarpeellisia jatkotoimenpiteitä.Kouvola Innovation Ltd is a limited liability company owned by City of Kouvola and it is a client of Kunnan Taitoa Ltd, which main line of business is combined office administrative service activities. Different persons have been in charge of accounting at Kouvola Innovation and there has been no ready layout or common pattern for financial statement or balance sheet book. There are differences in appearance and content between annual balance sheet books of different years. The objective of this thesis was to create a new balance sheet book layout for Kunnan Taitoa Ltd’s Kouvola office by improving previous balance sheet books. The layout would be used to assist the making of the financial statement of Kouvola Innovation. The purpose was to create a layout which could be used with small adjustments in financial statements of different companies and which would retrieve numbers of financial statements from group accounting program’s database. The content of financial statement and balance sheet book are discussed in the light of legislation, regulation, guidelines and literature on this thesis. The theory was written simultaneously with the layout compilation. The improvements to appearance and content were aimed to be found by studying previous balance sheet books and theory

Generation of appropriately matched wild-type and OPN knockout cell lines using CRISPR-Cas9 mediated gene editing.

<p><b>(A)</b> Schematic of traditional and modified CRISPR/Cas9 based gene editing protocols. <b>(B)</b> Schematic diagram of sgRNA targeting the <i>spp1</i> gene loci. Protospacer sequence is highlighted in red. Protospacer adjacent motif (PAM) sequences are presented in green. <b>(C)</b> Recovery rates, gene editing efficiency, and rate of homozygous targeting of the OPN gene in indicated subclones. <b>(D)</b> Western blot for OPN protein in MC-22 WT and edited clones (P16, P23, and P38) cultured in the presence or absence of brefeldin A (BFA). Expected multiple Osteopontin isoforms were detected between ~37–50 kD. A non-specific band was detected in each sample, indicated by “n.s”. <b>(E)</b> Concentration of murine osteopontin (mOPN) in 24-hr conditioned media from MC-50 WT and edited clones (MC-50-KO1 and MC-50-KO2). mOPN levels were normalized to final cell count. Osteopontin was undetected (ND) in conditioned media collected from both edited subclones. <b>(F)</b> Immunofluorescence cytochemical staining for mOPN (red) in MT-2 WT and a validated MT-2 OPN-KO clone. Nuclei are counterstained with hematoxylin (blue). Scale = 100 μm.</p

Phenotypic heterogeneity of McNeuA and Met-1 subclonal populations.

<p><b>(A)</b> Schematic of subclone derivation from breast cancer cell lines. <b>(B,C)</b> Phase contrast images of representative McNeuA (B) and Met-1 (C) subclones to demonstrate morphologic variability. Scale bars = 100 μm. <b>(D,E)</b> Concentration of murine osteopontin (mOPN; ng/ml per 10⁶ cells) in 24-hr conditioned media from McNeuA (MC) sublcones (D) and Met-1 (MT) subclones (E).</p

Phenotypic and functional heterogeneity of McNeuA and Met-1 breast cancer cells.

<p><b>(A)</b> Concentration of murine OPN (mOPN; ng/ml per 10⁶ cells) in 24-hr conditioned medium of McNeuA and Met-1 murine mammary carcinoma cells represented as mean ± SD. There was no detectable mOPN in the control cell-free medium (DMEM) (2 technical replicates per group). <b>(B)</b> Incidence of tumor formation following injection of indicated numbers of McNeuA or Met-1 cells into cohorts of FVB mice. <b>(C)</b> Plasma mOPN concentration (ng/ml) in indicated cohorts of mice at experimental end points of 84 days (McNeuA) and 30 days (Met-1). For McNeuA tumor-bearing mice, blue data points represent 10,000 cells injected, red data points represent 100,000 cells injected; n = 6–7 for McNeuA cohorts; n = 5–8 for Met-1 cohorts. Error bars represent SD; statistical significance evaluated using unpaired, two-tailed Student’s t-test. <b>(D)</b> Representative images of immunohistochemical staining for murine E-cadherin (red) on recovered McNeuA and Met-1 tumors. Cell nuclei were counterstained with hematoxylin (blue). Scale bars = 100 μm. (B-D) representative of 3 independent experiments per cell line. <b>(E)</b> Average radiance (log₁₀) per mouse (n = 5) as measured by bioluminescence imaging over 21-day time course following intravenous injection of 10⁶ Met-1 tumor cells into FVB mice (left graph). Fold-change (log₂) in pulmonary metastatic burden per mouse (right graph). Representative of 2 independent experiments. <b>(F)</b> Response of orthotopic Met-1 GFP/Luc tumors to single dose combination doxorubicin (5 mg/kg), paclitaxel (10 mg/kg) and cyclophosphamide (120 mg/kg) (AC-T), n = 5–8 tumors/group. Ordinate represents time (days) following treatment. Error bars represent SEM; two-way ANOVA Sidak’s multiple comparisons test; **p<0.01. Representative of 3 independent experiments. <b>(G)</b> Growth kinetics of individual orthotopic Met-1 Luc/GFP tumors in mice injected with 2.5 x 10⁵ tumor cells at the experiment initiation, subsequently receiving 4 biweekly AC-T doses (red arrows). Numbers and colors represent individual mice.</p

OPN depletion does not affect primary tumor formation in murine models of HER2⁺ and ER^- breast cancer.

<p><b>(A-C)</b> FVB mice were orthotopically injected with 10⁵ MC-22 (A), 10⁵ MC-50 (B), or 2.5 x 10⁴ MT-2 (C) cells. Growth kinetics (mm³) of orthotopic tumors of WT (blue lines) and validated OPN-KO clones (red lines). Mass of primary tumors from WT (blue) or OPN-KO (red) cohorts at experimental end points. No statistically significant differences were determined by 2way ANOVA (tumor growth kinetics) or unpaired, two-tailed Students’ t-test (tumor mass) statistical analyses. Circulating plasma murine osteopontin (mOPN) levels from cancer-free (green) or tumor bearing mice from the MC-22, MC-50, or MT-2 WT (blue) or OPN-KO (red) cohorts (One-way ANOVA: *** p = 0.0003, **** p < 0.0001). Error bars represent SD. <b>(D)</b> Representative immunohistochemical staining for mOPN (red) in tumors derived from MC-22, MC-50 and MT-2 WT and validated OPN-KO cell lines. Cell nuclei counterstained with hematoxylin (blue). Scale bar = 50 μm.</p

MT-2 OPN-KO derived tumors exhibit enhanced chemosensitivity <i>in vivo</i>.

<p><b>(A)</b> Experimental schema. 2.5 × 10⁴ MT-2 WT or OPN-KO tumor cells were injected into the mammary fat pads of 6–8-week-old female FVB mice. A single dose of AC-T was initiated at 14 days, when tumors reached ~60–80 mm³ in volume, and tumor growth was monitored periodically until the end point of 44 days. Error bars represent SD. <b>(B)</b> Tumor growth kinetics for MT-2 WT vehicle (blue; n = 5) and AC-T treated (green; n = 4) and MT-2 OPN-KO vehicle (red; n = 3) and AC-T treated mice (purple; n = 2). Representative of 3 biological repetitions. Error bars represent SD. <b>(C)</b> Endpoint tumor mass for MT-2 WT and MT-2 OPN-KO AC-T treated mice from 2 separate experiments (Mann-Whitney, p = 0.0037; endpoint tumor mass was not measured during the first of the three experimental repetitions). Data points from individual repetitions are represented with different colors. Error bars represent SD.</p

Matched wild type and knockout OPN cell lines can be used for pre-clinical metastasis studies.

<p><b>(A)</b> Experimental schema for metastasis assay. <b>(B)</b> Representative <i>in vivo</i> bioluminescent images of mice injected with MT-2 WT or MT-2 OPN KO after 7d and 21d. <b>(C)</b> Average fold change of bioluminescent signal (radiance (p/sec/cm²/sr), log10, normalized for differences in Luciferase expression between cell lines) from mice with MT-2 WT (blue) or MT-2 OPN KO (red) at indicated time points. (unpaired, two tailed t-test: *** p = 0.000067). Error bars represent SEM. <b>(D)</b> Representative hematoxylin & eosin staining of lungs from mice that received tail vein injections of MT-2 WT or MT-2 OPN KO cells. An example of a multifocal metastasis is marked with a blue arrow and an example of a single focus metastasis is marked with a red arrow. Scale = 1000 μm. <b>(E)</b> Quantification of total metastases in MT-2 WT (blue) and MT-2 OPN KO (red) cohorts (WT n = 21, KO n = 30; Mann-Whitney, p = 0.0466). Error bars represent SD. <b>(F)</b> Quantification of multifocal metastases in MT-2 WT (blue) and MT-2 OPN KO (red) cohorts (WT n = 21, KO n = 30; Mann-Whitney, p = 0.0185). Error bars represent SD.</p