Cell Cycle Progression Pathways Research Articles

Abstract B072: Loss of the tumor and metastasis suppressor RasGAP DAB2IP mediates therapeutic resistance in HER2+ breast cancer

Abstract Resistance to HER2 inhibitors remains a clinical challenge in HER2+ breast cancer. Therefore, there is an urgent need to 1) understand the mechanisms that underlie resistance to these current treatments and 2) develop improved, and more importantly, curative combination therapies. We previously showed that the RasGAP DAB2IP is a tumor and metastasis suppressor in breast cancer. Interestingly, we have now generated robust data demonstrating that the loss of DAB2IP also mediates therapeutic resistance in HER2+ breast cancer. First, we genetically ablated DAB2IP in multiple HER2+ breast cancer cell lines and performed manual counting experiments after 6 days of HER2 TKI treatment. In all cell lines, DAB2IP knockdown conferred resistance to HER2 inhibitors. Since HER2 inhibitors are known to induce both apoptosis and senescence, we next investigated how DAB2IP loss specifically affects these phenotypes. Interestingly, DAB2IP knockdown prevented TKI-induced caspase-3/7 activation, measured by Incucyte live cell imaging, and enabled the regrowth of cells in long-term 10-day treatment and drug washout experiments, monitored by Incucyte or crystal violet staining. Mechanistically, stable knockdown of DAB2IP reduced the suppression of the AKT and ERK pathways and the induction of the pro-apoptotic protein Bim upon HER2i treatment. In addition, cell cycle progression pathways were enriched in DAB2IP-deficient cells on lapatinib treatment and immunoblots revealed higher residual levels of pRb and low levels of p27 in these cells. Interestingly, the levels of these proteins were restored and regrowth in low density assays was inhibited with co-treatment with an NF-kB inhibitor. We next evaluated the relevance of DAB2IP loss in mediating HER2 inhibitor resistance in a SUM190 orthotopic xenograft model. Importantly, while control tumors regressed upon lapatinib treatment, DAB2IP-deficient tumors did not regress and grew with kinetics comparable to vehicle-treated tumors after few days on treatment. Further understanding the pathways deregulated by DAB2IP loss upon HER2i treatment will be essential for developing effective therapeutic strategies to combat resistance induced by DAB2IP loss. Citation Format: Naiara Perurena, Amy Schade, Natalie Pilla, Patrick Loi, Carrie Rodriguez, Alycia Gardner, Karen Cichowski. Loss of the tumor and metastasis suppressor RasGAP DAB2IP mediates therapeutic resistance in HER2+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B072.

Functional and molecular profiling of fasted piglets reveals decreased energy metabolic function and cell proliferation in the small intestine.

The small intestine requires energy to exert its important role in nutrient uptake and barrier function. Pigs are an important source of food and a model for humans. Young piglets and infants can suffer from periods of insufficient food intake. Whether this functionally affects the small intestinal epithelial cell (IEC) metabolic capacity and how this may be associated with an increased vulnerability to intestinal disease is unknown. We therefore performed a 48-h fasting intervention in young piglets. After feeding a standard weaning diet for 2 wk, 6-wk-old piglets (n = 16/group) were fasted for 48 h, and midjejunal IECs were collected upon euthanasia. Functional metabolism of isolated IECs was analyzed with the Seahorse XF analyzer and gene expression was assessed using RNA-sequencing. Fasting decreased the mitochondrial and glycolytic function of the IECs by 50% and 45%, respectively (P < 0.0001), signifying that overall metabolic function was decreased. The RNA-sequencing results corroborated our functional metabolic measurements, showing that particularly pathways related to mitochondrial energy production were decreased. Besides oxidative metabolic pathways, decreased cell-cycle progression pathways were most regulated in the fasted piglets, which were confirmed by 43% reduction of Ki67-stained cells (P < 0.05). Finally, the expression of barrier function genes was reduced upon fasting. In conclusion, we found that the decreased IEC energy metabolic function in response to fasting is supported by a decreased gene expression of mitochondrial pathways and is likely linked to the observed decreased intestinal cell proliferation and barrier function, providing insight into the vulnerability of piglets, and infants, to decreased food intake.NEW & NOTEWORTHY Fasting is identified as one of the underlying causes potentiating diarrhea development, both in piglets and humans. With this study, we demonstrate that fasting decreases the metabolism of intestinal epithelial cells, on a functional and transcriptional level. Transcriptional and histological data also show decreased intestinal cell proliferation. As such, fasting-induced intestinal energy shortage could contribute to intestinal dysfunction upon fasting.

Abstract P1-13-06: Loss of emerging tumor and metastasis suppressor RasGAPs mediates therapeutic resistance in HER2+ breast cancer

Abstract Resistance to HER2 inhibitors remains a clinical challenge in HER2+ breast cancer. Therefore, there is an urgent need to 1) understand the mechanisms that underlie resistance to these current treatments and 2) develop improved, and more importantly, curative combination therapies. We recently discovered that two emerging tumor suppressor RasGAPs, DAB2IP and RASAL2, cooperatively drive metastatic breast cancer when lost or inactivated. Interestingly, we have now generated robust data demonstrating that the loss of these RasGAPs also induces resistance to HER2 inhibitors in breast cancer. First, we genetically ablated both RASAL2 and DAB2IP in multiple HER2+ breast cancer cell lines (SKBR3, EFM192A, SUM190, BT474) and performed manual counting experiments after 6 days of TKI (lapatinib, tucatinib) treatment. In all cell lines, RASAL2/DAB2IP knockdown conferred resistance to HER2 inhibitors. Moreover, loss of these RasGAPs prevented TKI-induced caspase-3/7 activation, measured by Incucyte live cell imaging, and enabled the regrowth of cells in long-term 10-day treatment and drug washout experiments, monitored by Incucyte or crystal violet staining. Next, we sought to investigate the individual contribution of each RasGAP to these phenotypes. Surprisingly, we found that RASAL2 and DAB2IP functioned quite differently in this context. Specifically, while RASAL2 loss prevented apoptosis, DAB2IP loss prevented irreversible cell cycle arrest (measured by functional long-term experiments and EdU staining assays). Mechanistically, RASAL2 loss uniquely impaired BIM induction at both mRNA and protein levels, which is required for lapatinib-induced cell death of HER2+ cancer cells. By contrast, cell cycle progression pathways were uniquely enriched in DAB2IP-deficient cells on lapatinib treatment and immunoblots revealed higher residual levels of pRb and low levels of p27 in these cells. These data suggest that RASAL2 and DAB2IP (loss) mediate resistance to HER2 inhibitors by differentially deregulating unique pathways/phenotypes. We next evaluated the relevance of these findings in a SUM190 orthotopic xenograft model. Importantly, while control tumors (expressing both RASAL2 and DAB2IP) regressed upon lapatinib treatment, DAB2IP- and RASAL2-deficient tumors did not regress and grew with kinetics comparable to vehicle-treated tumors after few days on treatment. These data suggest that the unique phenotypes/pathways induced by both RASAL2 and DAB2IP are important mediators of resistance to HER2 inhibitors. Further understanding the contribution of these pathways to anti-HER2 resistance and determining how RASAL2 and DAB2IP differentially function will be essential for developing effective therapeutic strategies to bypass each type of resistance. Citation Format: Naiara Perurena, Natalie Pilla, Amy Schade, Marina Watanabe, Patrick Loi, Carrie L. Rodriguez, Alycia M. Gardner, Karen Cichowski. Loss of emerging tumor and metastasis suppressor RasGAPs mediates therapeutic resistance in HER2+ breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-06.

Genomic And Tumor Microenvironment Differences Between Cell Cycle Progression Pathway Altered/Non-Altered Patients With Lung Adenocarcinoma.

BackgroundIdentified as a hallmark of cancer, the dysregulated cell cycle progression plays an important role in the promotion and progression of lung adenocarcinoma (LUAD). However, the genomic and microenvironment differences between cell cycle progression pathway altered/non-altered LUAD patients remain to be elucidated.Materials and MethodsData of this study were obtained from The Cancer Genome Atlas (TCGA), including simple nucleotide variation, copy number variation (CNV), RNA-seq gene expression, miRNA expression, survival, and clinical information. Besides, 34 LUAD samples from our institution were used as a validation cohort. Differentially expressed genes (DEGs), enrichment analysis, and immune cell infiltration were detected. At last, we built a LASSO-binary Logistic regression model to predict the cell-cycle-related gene mutation (CDKN2A, CCND1, CDK4, CCNE1, and RB1) in LUAD patients and further verified it in the samples from our institution.ResultsBased on the cell cycle progression pathway status, the LUAD patients were divided into the mutation (n=322) and wild (n=46) groups. Compared to the wild group, the mutation group had a higher mutational load and CNV. Among the 16684 protein-coding genes analyzed, 302 were upregulated, and 354 were downregulated in the mutation group. Enrichment analysis indicated that these DEGs were closely related to metabolism items. After performing immune cell infiltration analysis of 22 immune cells, we found the proportion of 5 immune cells such as monocytes (P<0.01) and dendritic cells (P<0.01) were higher in the wild group. Finally, a cell-cycle-related 15-signature model was built by LASSO-Logistic regression analysis, which could predict the cell cycle progression pathway-related gene mutation (CDKN2A, CCND1, CDK4, CCNE1, and RB1) in LUAD patients. The validation cohorts showed the sensitivity and specificity of this model were 0.667 and 0.929, respectively.ConclusionThe genomic and microenvironment characteristics differed between the cell cycle progression pathway altered/non-altered patients with LUAD. Our findings may provide new insight into personalized treatment for LUAD patients.

Abstract P5-12-10: A commensal’s exopolysaccharide inhibits breast cancer proliferation in vitro but promotes tumor growth in vivo

Abstract Background: Recent decades have started to realize the importance of the microbiome in cancer initiation, progression and response to therapy. Recent findings suggest that both the local and distal microbiome may influence tumor growth. Although many studies focused on the gut microbiome, the breast itself also possesses a unique microbiome which differs between healthy and breast cancer patients, and between tumor subtypes. However, it is poorly understood whether these bacteria in the gut or the breast microenvironment may interact with breast cancer cells and if that interaction alters tumor growth. Bacteria produce metabolites and other signaling molecules to alter the host’s response. We have identified and purified an exopolysaccharide (EPS) produced by the commensal bacterium Bacillus subtilis, which was shown to act directly on the host’s innate immune cells to protect against various inflammatory diseases. This study aims to investigate if, and how the bacterial product (EPS) alters proliferation and tumor growth of breast cancer cells.Methods: Cell proliferation of bulk cells was conducted on PBS or EPS treated breast cancer cells (T47D, MDA-MB-468, MDA-MB-453, HCC1428, BT549, MDA-MB-231, ZR-75-30, and MCF-7) using trypan blue exclusion and the XTT assay. Flow cytometry was used to assess apoptosis with Annexin V, and cell cycle progression with propidium iodide. Cancer stem cell survival was assessed using the mammosphere forming assay. CRISPR/Cas9-mediated knockout of the Toll-like-receptor 4 (TLR4) was used to assess the requirement of TLR4 signaling on EPS-mediated phenotypes. RNA-sequencing was performed on both EPS-sensitive and resistant breast cancer cell lines after treatment with PBS or EPS for 20 hours. T47D xenografts studies were performed in vivo using EPS pretreatment and continual treatment via i.p. injection.Results: EPS inhibited proliferation of some breast cancer cell lines (T47D, MDA-MB-468, MDA-MB-453, HCC1428) in a concentration dependent manner while having little effect on others (BT549, MDA-MB-231, ZR-75-30, MCF7). EPS acted via distinct mechanisms depending on the cell line, increasing apoptosis in MDA-MB-468 cells while inducing G0/G1 cell cycle arrest in T47D cells. Although EPS directly inhibited bulk cell proliferation, the cancer stem cell population of T47D cells was increased by EPS. EPS is known to require TLR4 signaling to modulate immune cells. CRISPR/Cas9 knockout of TLR4 demonstrated that it was not required for EPS-mediated inhibition of T47D breast cancer cells, suggesting a novel mechanism of action in this cell line. RNA-sequence analysis showed that EPS altered interferon signaling and cell cycle progression pathways. Interferon signaling activates the STAT family of transcription factors, and western blots confirmed that 3h of EPS treatment induced STAT1 phosphorylation and p21 accumulation, indicating that EPS activated this pathway. Contrary to in vitro proliferation data, in vivo results showed that EPS-treated cells formed significantly larger tumors compared to PBS controls (273mg vs 59mg). The slope of tumor growth was also significantly faster in the EPS-treated group. Conclusions: These findings demonstrated the multifacet of EPS’s effect on breast cancer cells. EPS inhibits the proliferation of certain breast cancer cells in vitro, possibly via activation of STAT1 and p21 signaling, but also increases survival of cancer stem cells and promotes more aggressive tumor growth in vivo. This highlights the complexity of the influence of commensal bacteria on breast cancer growth, which can be highly context-dependent. Deeper understanding of this complex interplay between the host-microbe-tumor will be vital to the development of novel bacterial strategies in the future. Citation Format: Mai Rachel Nguyen, Katherine L Knight, Clodia Osipo. A commensal’s exopolysaccharide inhibits breast cancer proliferation in vitro but promotes tumor growth in vivo [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-10.

LRP1B and GRM3 expression in colorectal cancer.

177 Background: LRP1B is a member of the low-density lipoprotein receptor family and a tumor suppressor found to be downregulated in colon cancer (CRC). GRM3 is a receptor of glutamate, an amino acid and neurotransmitter. Inhibition of GRM3 reduces CRC cell growth. Recent data from CALGB/SWOG 80405 suggests that mutations (MT) of either LRP1B or GRM3 are associated with better and worse overall survival (OS) in patients treated with bevacizumab (Bev), respectively. We investigate the association of LRP1B or GRM3 mRNA levels with outcomes. Methods: A total of 13,780 CRC tumors (male 7,497, female 6,283) underwent comprehensive molecular profiling (Caris Life Sciences). Analyses included next-generation sequencing of DNA (592 genes, NextSeq, WES, NovaSEQ) and RNA (NovaSeq). Significance with multiple correction was indicated with q, otherwise p value. Gene Set Enrichment Analyses (GSEA) were performed (significance p &lt;.05). A Consensus Molecular Subtype (CMS) calling algorithm was developed using mRNA levels (transcripts per million; TPM). Time on treatment (TOT) with Bev was extracted from insurance claims. Results: Male patients had higher GRM3 expression (median TPM.55 vs..52, p &lt;.001). GRM3 and LRP1B were both elevated in brain metastases (1.95 vs..40, q&lt;.01;.53 vs..16, q&lt;.01) and enriched in CMS4 subtype (both p &lt;.001). Overexpression of GRM3 and LRP1B were significantly associated with MSS (.11 vs..07, p &lt;.0001;.54 vs..39, p &lt;.0001) and TMB low status (.11 vs..08, p &lt;.0001;.54 vs..40, p &lt;.0001). For MSS tumors, high LRP1B was associated with lower MT rates of APC (76% vs. 78%), KRAS (49% vs. 51%) and PIK3CA (15% vs. 17%). For MSI tumors, high LRP1B correlated with higher MT of MSH6 (41% vs. 32%), BRCA2 (28% vs. 20%) and PMS2 (12% vs. 6%). MSS tumors with high GRM3 had more APC (79% vs. 75%), less KRAS (47% vs. 52%) and SMAD4 (12% vs. 16%) MT and MSI with high GRM3 carried more APC (42% vs. 35%) and RAD50 (18% vs. 8%) MT. MSS tumors with low LRP1B showed upregulation of the EIF2 pathway while MTOR, RAB, and CDC42 pathways were enriched in MSI with low LRP1B. CDC42 and MTOR pathways were enriched in MSS tumors with low GRM3, and MSI with low GRM3 displayed enrichment of EIF2 and Notch pathways. In MSS tumors, both LRP1B and GRM3 were prognostic and associated with better survival (HR.66, 95% CI [.56-.78], p &lt;.0001 for LRP1B; HR.79, 95% CI [.68-.92], p &lt;.01 for GRM3) and high expression of either one was also associated with better prognosis for patients treated with Bev (HR.85, 95% CI [.70-.92], p &lt;.01 for LRP1B; HR 0.88, 95% CI [.77-.99], p &lt;.05 for GRM3). Conclusions: LRP1B and GRM3 appear to be important regulators in CRC because of their prognostic value and association with response to bevacizumab treatment. Both LRP1B and GRM3 are associated with pathways of cell cycle progression, cell migration, and DNA repair. A better understanding of their role in angiogenic signaling is critical to develop more effective strategies to improve response to bevacizumab or immunotherapy.

The Dual Effect of the HDAC Inhibitor Romidepsin on Androgen Receptor Signaling and DNA Damage Repair in Prostate Cancer.

Aberrant epigenetic modifications play an important role in prostate cancer development and progression. Histone deacetylase inhibitors (HDACi) are a novel class of small-molecular compounds that have been shown to have activity in several tumor types. Some HDACi's induce a stress response that affects DNA damage repair (radiosensitization) and androgen receptor (AR) signaling (increased sensitivity to androgen blockage). We hypothesized that the HDACi, romidepsin, would increase prostate cancer cell killing through dual effects on AR signaling and radiation DNA damage response.The prostate cancer cell lines LNCaP (androgen sensitive) and 22Rv1 (androgen insensitive) were treated with romidepsin (10 nM) plus or minus 2 Gy radiation. The added effect of enzalutamide (10 mM) was also tested. Cell viability was performed by Real-time Cell assay (RTCA), whole-transcriptome analysis by RNA sequencing, western blot analyses, immunofluorescent quantification of γ-H2AX double strand break foci, and colonogenic survival. Western blot analysis was used to measure the expression of (i) AR signaling pathway proteins that included wild-type AR and its AR-V7 splice variant and (ii) DNA repair genes that included Rad51, DNA-PK and γ-H2AX.Romidepsin reduced cell viability by RTCA of both cell lines in a dose dependent manner. The addition of enzalutamide further reduced cell viability and clonogenic survival. Gene ontology analysis from RNA-Seq data in both LNCaP and 22Rv1 cells revealed that romidepsin suppressed pathways related to prostate cancer aggressiveness including the AR, MYC, DNA damage response, and cell cycle progression pathways. By western blot analysis, romidepsin suppressed AR (LNCaP) and AR-V7 (22Rv1) expression and reduced DNA repair genes including RAD51, DNA-PK and phospho-ATM levels. After 2 Gy radiation, we found that treatment with 10nM romidepsin for 24 hours resulted in an increase in γ-H2AX foci in 35.2% in LNCaP and 27% in 22Rv1 cells respectively. By colonogenic assay, 10 nM romidepsin for 24 hours prior to 2 Gy to 8 Gy radiation resulted in a significant reduction in the surviving fractions. The addition of enzalutamide resulted in the greatest reduction in colonogenic survival.Our study demonstrated for the first time that HDACi romidepsin exerted dual effects on AR signaling and DNA damage response genes in androgen sensitive and insensitive prostate cancer cell lines, and that there is an added suppressive effect with the addition of enzalutamide to HDAC inhibition plus radiation. The use of HDACi's have promise clinically for the treatment of high-risk prostate cancer, for which androgen deprivation plus radiotherapy are the standard treatments.

Transcriptomic Analysis Reveals the MicroRNAs Responsible for Liver Regeneration Associated With Mortality in Alcohol-Associated Hepatitis.

We conducted a comprehensive serum transcriptomic analysis to explore the roles of microRNAs (miRNAs) in alcohol-associated hepatitis (AH) pathogenesis and their prognostic significance. Serum miRNA profiling was performed in 15 controls, 20 heavy drinkers without liver disease, and 65 patients with AH and compared to publicly available hepatic miRNA profiling in AH patients. Among the top 26 miRNAs, expression of miR-30b-5p, miR-20a-5p, miR-146a-5p, and miR-26b-5p were significantly reduced in both serum and liver of AH patients. Pathway analysis of the potential targets of these miRNAs uncovered the genes related to DNA synthesis and cell-cycle progression pathways, including ribonucleotide reductase regulatory subunit M2 (RRM2), cyclin D1 (CCND1), cyclin D2 (CCND2), MYC proto-oncogene (MYC), and phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1). We found a significant increase in the protein expression of RRM2, CCND1, and CCND2, but not MYC and PMAIP1, in AH patients who underwent liver transplantation; miR-26b-5p and miR-30b-5p inhibited the 3'-UTR (untranslated region) luciferase activity of RRM2 and CCND2, and miR-20a-5p reduced the 3'-UTR luciferase activity of CCND1 and CCND2. During a median follow-up of 346days, 21% of AH patients died; these patients had higher body mass index (BMI), Model for End-Stage Liver Disease (MELD), and serum miR-30b-5p, miR-20a-5p, miR-146a-5p, and miR-26b-5p than those who survived. Cox regression analysis showed that BMI, MELD score, miR-20a-5p, miR-146a-5p, and miR-26b-5p predicted mortality. Patients with AH attempt to deal with hepatocyte injury by down-regulating specific miRNAs and up-regulating genes responsible for DNA synthesis and cell-cycle progression. Higher expression of these miRNAs, suggestive of a diminished capacity in liver regeneration, predicts short-term mortality in AH patients.

Abstract 744: Progesterone receptors promote quiescence &amp; ovarian cancer cell phenotypes via modulation of DREAM in p53-mutant fallopian tube models

Abstract The ability of ovarian steroids to modulate ovarian cancer (OC) risk remains controversial. High grade serous ovarian carcinoma (HGSC), the most aggressive OC subtype, contains abundant estrogen (ER; 76%) and progesterone (PR; 35%) receptors. Progesterone is thought to be a primarily protective factor that reduces OC risk but the relevance of PR actions in HGSC is unknown. Our IHC analyses of human clinical tissues showed robust expression of total and activated phospho-S294 PR within serous tubal intraepithelial carcinomas (STIC), precursor HGSC lesions of fallopian tube epithelia (FTE), suggesting a role for PR signaling in early disease. To explore the molecular mechanisms of PR isoform actions, p53-dominant negative mutant FTE lines expressing the PR-A or PR-B isoform were created. Progestin treatment (R5020 or progesterone) of these models induced spheroid formation and reaggregation, transwell migration and collagen invasion. RNAseq analyses revealed enrichment of cell cycle progression pathways; in particular, the gene targets of the repressive DREAM complex that drives quiescent cell fate. Progestins induced inhibition of proliferation (decreased Ki67; decreased BrdU; increased B-galactosidase) and accumulation of cells in G0/G1 that was reversed following growth factor stimulation. Subsequent immunoprecipitation of DREAM (G0) and B-MYB/MMB complexes (G1/S) confirmed that PR-driven changes in cell fate occur via regulation of the DREAM complex. PR activation enhanced DREAM and suppressed B-MYB/MMB complex formation. PR also induced transcriptional regulation of DREAM/B-MYB/MMB genes (LIN9, MYBL2, DYRK1A, FOXM1) via promoter recruitment and mRNA regulation. Interestingly, DREAM complexes contained PR and PR was co-recruited to DREAM binding sites within DREAM target promoters, along with the DREAM complex proteins p130 and E2F4. The relevance of the quiescent state to early OC phenotypes was examined through the disruption of DREAM/DYRK1s by pharmacological inhibition (harmine) , HPV E6/E7 expression or the depletion of DYRK1A/B kinases that promote DREAM complex formation. These manipulations blocked progestin-induced cell cycle arrest and attenuated PR-driven gene expression and associated OC phenotypes. Our results reveal that activated PRs support quiescence and pro-survival/pro-dissemination cell behaviors that may contribute to early HGSC progression. Taken together, our data suggest an alternative perspective on the tenant that progesterone always confers protection against OC. STICs can reside undetected for decades prior to invasive disease. Our studies reveal clinical opportunities to prevent the ultimate development of HGSC by targeting activated PRs, DREAM complexes, and/or DYRKs (Supported by the Minnesota Ovarian Cancer Alliance to LJM and CAL). Citation Format: Laura J. Mauro, Megan I. Seibel, Caroline H. Diep, Angela Spartz, Carlos Perez Kerkvliet, Hari Singhal, Elizabeth M. Swisher, Lauren E. Schwartz, Ronny Drapkin, Siddharth Saini, Fatmata Seesay, Larisa Litovchick, Carol A. Lange. Progesterone receptors promote quiescence &amp; ovarian cancer cell phenotypes via modulation of DREAM in p53-mutant fallopian tube models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 744.

Proteomic analysis of transitional cell carcinoma–like variant of tubo-ovarian high-grade serous carcinoma

The current World Health Organization classification does not distinguish transitional cell carcinoma of the ovary (TCC) from conventional tubo-ovarian high-grade serous carcinoma (HGSC), despite evidence suggesting improved prognosis for patients with TCC; instead, it is considered a morphologic variant of HGSC. The immunohistochemical (IHC) markers applied to date do not distinguish between TCC and HGSC. Therefore, we sought to compare the proteomic profiles of TCC and conventional HGSC to identify proteins enriched in TCC. Prognostic biomarkers in HGSC have proven to be elusive, and our aim was to identify biomarkers of TCC as a way of reliably and reproducibly identifying patients with a favorable prognosis and better response to chemotherapy compared with those with conventional HGSC. Quantitative global proteome analysis was performed on archival material of 12 cases of TCC and 16 cases of HGSC using SP3 (single-pot, solid phase–enhanced, sample preparation)-Clinical Tissue Proteomics, a recently described protocol for full-proteome analysis from formalin-fixed paraffin-embedded tissues. We identified 430 proteins that were significantly enriched in TCC over HGSC. Unsupervised co-clustering perfectly distinguished TCC from HGSC based on protein expression. Pathway analysis showed that proteins associated with cell death, necrosis, and apoptosis were highly expressed in TCCs, whereas proteins associated with DNA homologous recombination, cell mitosis, proliferation and survival, and cell cycle progression pathways had reduced expression. From the proteomic analysis, three potential biomarkers for TCC were identified, claudin-4 (CLDN4), ubiquitin carboxyl-terminal esterase L1 (UCHL1), and minichromosome maintenance protein 7 (MCM7), and tested by IHC analysis on tissue microarrays. In agreement with the proteomic analysis, IHC expression of those proteins was stronger in TCC than in HGSC (p < 0.0001). Using global proteomic analysis, we are able to distinguish TCC from conventional HGSC. Follow-up studies will be necessary to confirm that these molecular and morphologic differences are clinically significant.

Retraction Note: Increased survival and cell cycle progression pathways are required for EWS/FLI1-induced malignant transformation

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The characteristics of vessel lining cells in normal spleens and their role in the pathobiology of myelofibrosis

AbstractThe CD34−CD8α+, sinusoid lining, littoral cells (LCs), and CD34+CD8α−, splenic vascular endothelial cells (SVECs) represent 2 distinct cellular types that line the vessels within normal spleens and those of patients with myelofibrosis (MF). To further understand the respective roles of LCs and SVECs, each was purified from normal and MF spleens, cultured, and characterized. Gene expression profiling indicated that LCs were a specialized type of SVEC. LCs possessed a distinct gene expression profile associated with cytoskeleton regulation, cellular interactions, endocytosis, and iron transport. LCs also were characterized by strong phagocytic activity, less robust tube-forming capacity and a limited proliferative potential. These characteristics underlie the role of LCs as cellular filters and scavengers. Although normal LCs and SVECs produced overlapping as well as distinct hematopoietic factors and adhesion molecules, the gene expression profile of MF LCs and SVECs distinguished them from their normal counterparts. MF SVECs were characterized by activated interferon signaling and cell cycle progression pathways and increased vascular endothelial growth factor receptor, angiopoietin-2, stem cell factor, interleukin (IL)-33, Notch ligands, and IL-15 transcripts. In contrast, the transcription profile of MF LCs was associated with mitochondrial dysfunction, reduced energy production, protein biosynthesis, and catabolism. Normal SVECs formed in vitro confluent cell layers that supported MF hematopoietic colony formation to a greater extent than normal colony formation. These data provide an explanation for the reduced density of LCs observed within MF spleens and indicate the role of SVECs in the development of extramedullary hematopoiesis in MF.

Increased survival and cell cycle progression pathways are required for EWS/FLI1-induced malignant transformation.

Ewing sarcoma (ES) is the second most frequent childhood bone cancer driven by the EWS/FLI1 (EF) fusion protein. Genetically defined ES models are needed to understand how EF expression changes bone precursor cell differentiation, how ES arises and through which mechanisms of inhibition it can be targeted. We used mesenchymal Prx1-directed conditional EF expression in mice to study bone development and to establish a reliable sarcoma model. EF expression arrested early chondrocyte and osteoblast differentiation due to changed signaling pathways such as hedgehog, WNT or growth factor signaling. Mesenchymal stem cells (MSCs) expressing EF showed high self-renewal capacity and maintained an undifferentiated state despite high apoptosis. Blocking apoptosis through enforced BCL2 family member expression in MSCs promoted efficient and rapid sarcoma formation when transplanted to immunocompromised mice. Mechanistically, high BCL2 family member and CDK4, but low P53 and INK4A protein expression synergized in Ewing-like sarcoma development. Functionally, knockdown of Mcl1 or Cdk4 or their combined pharmacologic inhibition resulted in growth arrest and apoptosis in both established human ES cell lines and EF-transformed mouse MSCs. Combinatorial targeting of survival and cell cycle progression pathways could counteract this aggressive childhood cancer.

UCH-L1 is induced in germinal center B cells and identifies patients with aggressive germinal center diffuse large B-cell lymphoma

AbstractGene expression profiling has identified 2 major subclasses of diffuse large B-cell lymphoma (DLBCL). Cases resembling germinal center (GC) B cells (GCB-DLBCL) generally occur in younger patients, have a distinct molecular pathophysiology, and have improved outcomes compared with those similar to activated post-GC cells (activated B-cell DLBCL). We previously found that the ubiquitin hydrolase UCH-L1 is frequently overexpressed in mature B-cell malignancies and is a potent oncogene in mice. The cause for its overexpression in lymphoma, and whether it impacts the outcome of patients with DLBCL is unknown. Here, we show that UCH-L1 reflects GC lineage in lymphoma and is an oncogenic biomarker of aggressive GCB-DLBCL. We find that UCH-L1 is specifically induced in GC B cells in mice and humans, and that its expression correlates highly with the GCB subtype in DLBCL. We also find that UCH-L1 cooperates with BCL6 in a mouse model of GC B-cell lymphoma, but not with the development of multiple myeloma derived from post-GC cells. Despite the typically good outcomes of GCB-DLBCL, increased UCHL1 identifies a subgroup with early relapses independent of MYC expression, suggesting biological diversity in this subset of disease. Consistent with this, forced Uchl1 overexpression had a substantial impact on gene expression in GC B cells including pathways of cell cycle progression, cell death and proliferation, and DNA replication. These data demonstrate a novel role for UCH-L1 outside of the nervous system and suggest its potential use as a biomarker and therapeutic target in DLBCL.

3,39-Diindolylmethane Ameliorates Staphylococcal Enterotoxin B–Induced Acute Lung Injury through Alterations in the Expression of MicroRNA that Target Apoptosis and Cell-Cycle Arrest in Activated T Cells.

3,39-Diindolylmethane (DIM), a natural indole found in cruciferous vegetables, has significant anti-cancer and anti-inflammatory properties. In this current study, we investigated the effects of DIM on acute lung injury (ALI) induced by exposure to staphylococcal enterotoxin B (SEB). We found that pretreatment of mice with DIM led to attenuation of SEB-induced inflammation in the lungs, vascular leak, and IFN-g secretion. Additionally, DIM could induce cell-cycle arrest and cell death in SEB-activated T cells in a concentration-dependent manner. Interestingly, microRNA (miRNA) microarray analysis uncovered an altered miRNA profile in lung-infiltrating mononuclear cells after DIM treatment of SEB-exposed mice. Moreover, computational analysis of miRNA gene targets and regulation networks indicated that DIM alters miRNA in the cell death and cell-cycle progression pathways. Specifically, DIM treatment significantly downregulated several miRNA and a correlative increase associated gene targets. Furthermore, overexpression and inhibition studies demonstrated that DIM-induced cell death, at least in part, used miR-222. Collectively, these studies demonstrate for the first time that DIM treatment attenuates SEB-induced ALI and may do so through the induction of microRNAs that promote apoptosis and cell-cycle arrest in SEB-activated T cells.

UCH-L1 Cooperates with BCL6 and Identifies Patients with Aggressive Germinal Center Diffuse Large B-Cell Lymphoma

Gene expression profiling has identified two major subclasses of diffuse large B-cell lymphoma. Cases resembling germinal center B-cells (GCB-DLBCL) generally occur in younger patients, have a distinct molecular pathophysiology, and have improved outcomes compared with those similar to activated post-germinal center cells (ABC-DLBCL). We previously found that the ubiquitin hydrolase UCH-L1 is frequently overexpressed in mature B-cell malignancies and is a potent oncogene in mice. The cause for its overexpression in lymphoma, and whether it impacts the outcome of patients with DLBCL is unknown. Here we show that UCH-L1 reflects germinal center lineage in lymphoma and is an oncogenic biomarker of aggressive GCB-DLBCL. We find that UCH-L1 is specifically induced in germinal center B-cells in mice and humans, and that its expression correlates highly with the GCB subtype in DLBCL. Despite the typically good outcomes of GCB-DLBCL, increased UCHL1 identifies a subgroup with early relapses independent of MYC expression, suggesting biologic diversity in this subset of disease. We also find that UCH-L1 synergizes with BCL6 in a mouse model of germinal center B-cell lymphoma, but not with the development of multiple myeloma derived from post-germinal center cells. Consistent with this, forced Uchl1 overexpression had a substantial impact on gene expression in germinal center B-cells including pathways of cell cycle progression, cell death and proliferation, and DNA replication. These data demonstrate a novel role for UCH-L1 outside of the nervous system and suggest its potential use as a biomarker and therapeutic target in DLBCL. DisclosuresGalardy:Mission Therapeutics: Research Funding.

Role of Centrosome in Regulating Immune Response

Centrosomes are the vital component of cell cycle progression pathway. Recent investigations have suggested their role in regulating the immune response system. Centrosome polarization delivers secretory granules to the immunological synapse (IS). The Cytotoxic T lymphocytes use a specific mechanism, controlled by centrosome delivery to the plasma membrane for delivering the secretory granules to the immunological synapse. Moreover, the polarization of centrioles to the immunological synapse directs secretion from cytolytic cells of innate as well as adaptive immune systems. Although the recent investigations have suggested their strong role in mediating the crucial events of immunological response, there are few discrepancies that are yet to be resolved. Furthermore, a clear picture of their molecular mechanism along with their cellular functions has not been reported. In this manuscript we have reviewed some important points that explain the importance of centrosomes in mediating the immunological signals and the delivery of lytic discharge from the cytotoxic and killer cells.

Could Upregulated Hsp70 Protein Compensate for the Hsp90-Silence-Induced Cell Death in Glioma Cells?

The molecular chaperone heat shock protein 90 alpha (Hsp90α) has been recognized in various tumours including glioma. This pilot study using a proteomic approach analyses the downstream effects of Hsp90 inhibition using 17-allylamino-17-demethoxygeldanamycin (17AAG) and a short hairpin RNA (shRNA) oligonucleotide targeting hsp90α (shhsp90α) in the U87-MG glioma cell line. Preliminary data coupled with bioinformatic analysis identified several known and unknown Hsp90 client proteins that demonstrated a change in their protein expression after Hsp90 inhibition, signifying an alteration in the canonical pathways of cell cycle progression, apoptosis, cell invasion, angiogenesis, and metastasis. Members of the glycolysis pathway were upregulated, demonstrating increased dependency on glycolysis for energy source by the treated glioma cells. Upregulated proteins also include Hsp70 and members of its family such as Hsp27 and gp96, thereby suggesting the role of Hsp90 co-chaperones in compensating for Hsp90 function after Hsp90 inhibition. Considering Hsp70’s role in antiapoptosis, it was postulated that a combination therapy involving a multitarget approach could be carried out. Consequently inhibition of both Hsp90 and Hsp70 in U87-MG glioma cells resulted in 60% cell death indicating the importance of combination therapy for glioma therapeutics.

Renoprotective mechanisms of telmisartan on renal injury and inflammation in SHRSP.Z-Leprfa/IzmDmcr rats

SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP fatty) rats create a new animal model of metabolic syndrome. However, the renoprotective effect of telmisartan therapy and its underlying mechanisms in SHRSP fatty rats remain unknown. We evaluate the effects of long-term telmisartan therapy on renal dysfunction, podocyte injury, inflammation, and transforming growth factor-β1 (TGF-β1)/Smad, epithelial-mesenchymal transition (EMT), mitogen-activated protein kinase (MAPK), Rho-kinase, and cell-cycle progression pathway in the renal cortex of SHRSP fatty rats. Seven-week-old male SHRSP fatty rats were treated with vehicle, telmisartan, and hydralazine for 8 weeks. Age-matched male Wistar-Kyoto/Izumo rats served as a control group. Vehicle-treated SHRSP fatty rats developed proteinuria and renal dysfunction, which in the telmisartan group was less than the vehicle and hydralazine group without changing blood pressure. Glomerulosclerosis and interstitial fibrosis were impaired in SHRSP fatty rats, and the renal damage in the telmisartan group was less than the vehicle and hydralazine groups. Decreased expression of nephrin and podocin and increased desmin-positive area in SHRSP fatty rats were restored by telmisartan but not hydralazine. TGF-β1/Smad, EMT marker, MAPK, Rho-kinase, and cell-cycle progression pathways were upregulated in SHRSP fatty rats, and these increased proteins in the telmisartan group were less than the vehicle and hydralazine group. Telmisartan administration resulted in significant suppression in tumor necrosis factor-α expression and nuclear factor-κB phosphorylation. Long-term telmisartan therapy may improve renal dysfunction, glomerulosclerosis, podocyte injury, and inflammation associated with EMT, TGF-β/Smad, MAPK, Rho-kinase pathway in SHRSP fatty rats. Thus, telmisartan may have significant therapeutic potential for metabolic syndrome.

Potential effects of CRM1 inhibition in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin lymphoma. The disease has no known cure, which prompts the urgent need for novel therapeutic agents. Chromosomal region maintenance 1 (CRM1) may play a role in human neoplasia and serve as a novel target of cancer treatment. This study summarizes MCL pathogenesis and determines the involvement of CRM1 in the regulation of several vital signaling pathways contributing to MCL pathogenesis, including the pathways of cell cycle progression, DNA damage response, phosphoinositide kinase-3, nuclear factor-κB activation, and chromosomal stability. A preclinical study is also presented to compare the CRM1 status in MCL cell lines and primary MCL cells with normal B cells, as well as the therapeutic efficiency of CRM1 inhibition in MCL in vitro and in vivo, which make these agents potential targets of novel MCL treatments.