The Function and Regulation of Senescent Stromal-Derived Osteopontin

Our understanding of tumors as complex organs has increased our appreciation for each component of the tumor microenvironment and its respective contribution to tumorigenesis. Fibroblasts found within a tumor actively participate in the growth, progression and metastasis of cancer cells. Senescent fibroblasts, which are permanently arrested yet metabolically active, accumulate in tissue over time where they may promote the proliferation and malignant conversion of preneoplastic cells in older individuals. I have examined the relationship between senescent fibroblasts and preneoplastic keratinocytes and identified the secreted multifunctional protein osteopontin: OPN) as a critical stromal mediating factor. RNAi-directed reduction of stromal OPN leads to decreased growth of preneoplastic keratinocytes in vitro and in vivo. Furthermore, I have demonstrated the presence of senescent stroma and associated OPN expression in the early stages of a chemical carcinogenesis mouse model, suggesting that it plays a role early in tumor formation. OPN is part of a unique transcriptional profile activated upon senescence induction. Given the significance of senescent stromal-derived factors in tumorigenesis, I have investigated the regulation of OPN and other members of the senescence-associated secretory profile: SASP). I demonstrate that SASP is coordinately upregulated in response to multiple stimuli inducing senescence namely DNA damage, chromatin modulation and ectopic expression of p27, a cyclin-dependent kinase inhibitor. However, my work on the transcriptional activation of OPN reveals that SASP contains distinct subsets governed by unique transcriptional mechanisms. To this end, I demonstrate that OPN, unlike IL6 and IL8 - two well characterized SASP members - does not require the activity of ATM or NfκB for its expression in senescence. To uncover specific activators of OPN transcription, I examined OPN\u27s promoter and I identified a fragment that is responsive in senescent fibroblasts. Furthermore, I demonstrate that OPN transcription requires chromatin modulators. Together my work presents a model where chromatin modifications in senescence play a central role in dictating transcriptional responses that ultimately impact the tumor microenvironment

Histone Acetylation-Mediated Regulation of the Hippo Pathway

The Hippo pathway is a signaling cascade recently found to play a key role in tumorigenesis therefore understanding the mechanisms that regulate it should open new opportunities for cancer treatment. Available data indicate that this pathway is controlled by signals from cell-cell junctions however the potential role of nuclear regulation has not yet been described. Here we set out to verify this possibility and define putative mechanism(s) by which it might occur. By using a luciferase reporter of the Hippo pathway, we measured the effects of different nuclear targeting drugs and found that chromatin-modifying agents, and to a lesser extent certain DNA damaging drugs, strongly induced activity of the reporter. This effect was not mediated by upstream core components (i.e. Mst, Lats) of the Hippo pathway, but through enhanced levels of the Hippo transducer TAZ. Investigation of the underlying mechanism led to the finding that cancer cell exposure to histone deacetylase inhibitors induced secretion of growth factors and cytokines, which in turn activate Akt and inhibit the GSK3 beta associated protein degradation complex in drug-affected as well as in their neighboring cells. Consequently, expression of EMT genes, cell migration and resistance to therapy were induced. These processes were suppressed by using pyrvinium, a recently described small molecule activator of the GSK 3 beta associated degradation complex. Overall, these findings shed light on a previously unrecognized phenomenon by which certain anti-cancer agents may paradoxically promote tumor progression by facilitating stabilization of the Hippo transducer TAZ and inducing cancer cell migration and resistance to therapy. Pharmacological targeting of the GSK3 beta associated degradation complex may thus represent a unique approach to treat cancer. © 2013 Basu et al

Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence

Additional file 3: Figure S3. Regulation of genes of Arrhythmogenic right ventricular cardiomyopathy pathway during senescence induction in HFF strains Genes of the “Arrhythmogenic right ventricular cardiomyopathy” pathway which are significantly up- (green) and down- (red) regulated (log2 fold change >1) during irradiation induced senescence (120 h after 20 Gy irradiation) in HFF strains. Orange color signifies genes which are commonly up-regulated during both, irradiation induced and replicative senescence

Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor–positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor–positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. Significance: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors

Senescence and aging – causes, consequences, and therapeutic avenues

Aging is the major risk factor for cancer, cardiovascular disease, diabetes, and neurodegenerative disorders. Although we are far from understanding the biological basis of aging, research suggests that targeting the aging process itself could ameliorate many age-related pathologies. Senescence is a cellular response characterized by a stable growth arrest and other phenotypic alterations that include a proinflammatory secretome. Senescence plays roles in normal development, maintains tissue homeostasis, and limits tumor progression. However, senescence has also been implicated as a major cause of age-related disease. In this regard, recent experimental evidence has shown that the genetic or pharmacological ablation of senescent cells extends life span and improves health span. Here, we review the cellular and molecular links between cellular senescence and aging and discuss the novel therapeutic avenues that this connection opens

Abstract A146: Preclinical models relevant to the treatment of post-menopausal breast cancer.

Abstract The past decades have witnessed immense progress in the treatment of hormone-sensitive breast cancer. However, despite the success of agents targeting estrogen activity, a significant proportion of patients will experience relapse due to endocrine therapy resistance. In order to better understand the underlying causes and enable successful targeting of implicated pathways, preclinical models that recapitulate the post-menopausal setting are essential. To this end, we have developed a panel of estrogen receptor (ER+) models with varying senstivity to endocrine therapies. Our panel includes models of long-term estrogen-deprivation (LTED), a surrogate for longterm exposure and relapse on aromatase inhibitors. Using both xenograft (MCF7LTED, HCC1428LTED) and primary tumor models (BR0555fLTED and CTC-174LTED), we have utilized them to benchmark standard of care treatments and test the efficacy of novel agents. Importantly, we demonstrate that these models closely mimick the post-menopausal hormonal millieu found in patients and recapitulate pharmacodynamic responses observed in the clinic. As such, these are instrumental tools to uncovering endocrine resistance mechanisms and provide a potentially predictive platform for clinical efficacy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A146. Citation Format: Ermira Pazolli, Anne Marie Mazzola, Jon Curwen, Hazel Weir, Casey Kiessel, Michael Grondine, Celina D'Cruz, Mike Zinda. Preclinical models relevant to the treatment of post-menopausal breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A146.</jats:p

Supplementary Figure 1 from Osteopontin Stimulates Preneoplastic Cellular Proliferation Through Activation of the MAPK Pathway

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