Retinoblastoma Tumor Suppressor Protein Research Articles

Secretory leukocyte protease inhibitor (SLPI) as a potential target for inhibiting metastasis of triple-negative breast cancers.

SLPI has been implicated in the progression and metastasis of certain cancers. However, the effects of SLPI seem to be tumor-specific and the mechanisms remain poorly defined. Here, we demonstrate that highly metastatic, triple-negative breast cancer (TNBC) 4T1 cells secreted more SLPI compared to their non-metastatic counterparts. Furthermore, SLPI secretion directly correlated with spontaneous lung metastasis from 4T1 tumors orthotopically implanted in mice. Consistent with our experimental results, we also found that higher SLPI expression levels correlate with worse clinical outcome in basal/TNBC patients. Using high-throughput screening we identified a novel compound, C74, which significantly inhibits SLPI secretion. C74 administration in our mouse model slows the growth of primary 4T1 tumors and inhibits their dissemination to the lung. We also discovered that SLPI physically interacts with the retinoblastoma tumor suppressor protein (Rb) and releases FoxM1 from the Rb-FoxM1 complex, which may activate FoxM1 target genes involved in breast cancer metastasis.

Hematological adverse effects in breast cancer patients treated with cyclin-dependent kinase 4 and 6 inhibitors: a systematic review and meta-analysis.

The introduction of specific cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly improved progression-free survival in hormone receptor-positive metastatic breast cancer. CDK 4/6 inhibitors induce cell cycle arrest via liberating the tumor suppressor retinoblastoma protein from CDK4/6 inhibitory effect. Preliminary studies suggested an increase in the hematological toxicities which might affect the quality of life in such palliative setting. We searched PubMed, ASCO, ESMO and San Antonio meeting databases for randomized phase II/III trials in metastatic breast cancer receiving CDK4/6 inhibitors with safety data provided on the incidence of hematological adverse effects. Our search identified 1012 citations that were screened for relevance. Thirty-six studies were found to be potentially eligible. After excluding the ineligible studies, six studies were deemed to be eligible for meta-analysis. The risk ratio (RR) was 11.31 [95% confidence interval (CI) 8.06-15.87; p<0.0001] for all-grade leucopenia, 14.86 (95% CI 11.37-19.41; p<0.0001) for all-grade neutropenia, 9.04 (95% CI 3.78-21.63; p<0.0001) for all-grade thrombocytopenia and 3.57 (95% CI 2.65-4.81; p<0.0001) for all-grade anemia. The RR for grade 3/4 leucopenia was 33.86 (95% CI 14.59-78.57; p<0.0001), for grade 3/4 neutropenia was 44.00 (95% CI 24.72-78.33; p<0.0001), for grade 3/4 thrombocytopenia was 5.70 (95% CI 2.03-16.01; p=0.001) and for grade 3/4 anemia was 2.80 (95% CI 1.45-5.41; p=0.002). There was no significant increase in the RR of febrile neutropenia with RR of 3.29 (95% CI 0.93-11.57; p=0.06). Our analysis provides evidence that the use of CDK 4/6 inhibitors is associated with an increased risk of all-grade and high-grade hematological adverse events, which seems to be a class-effect, but not of febrile neutropenia compared with hormonal therapy alone.

Tudor-domain protein PHF20L1 reads lysine methylated retinoblastoma tumour suppressor protein

The retinoblastoma tumour suppressor protein (pRb) classically functions to regulate early cell cycle progression where it acts to enforce a number of checkpoints in response to cellular stress and DNA damage. Methylation at lysine (K) 810, which occurs within a critical CDK phosphorylation site and antagonises a CDK-dependent phosphorylation event at the neighbouring S807 residue, acts to hold pRb in the hypo-phosphorylated growth-suppressing state. This is mediated in part by the recruitment of the reader protein 53BP1 to di-methylated K810, which allows pRb activity to be effectively integrated with the DNA damage response. Here, we report the surprising observation that an additional methylation-dependent interaction occurs at K810, but rather than the di-methyl mark, it is selective for the mono-methyl K810 mark. Binding of the mono-methyl PHF20L1 reader to methylated pRb occurs on E2F target genes, where it acts to mediate an additional level of control by recruiting the MOF acetyltransferase complex to E2F target genes. Significantly, we find that the interplay between PHF20L1 and mono-methyl pRb is important for maintaining the integrity of a pRb-dependent G1–S-phase checkpoint. Our results highlight the distinct roles that methyl-lysine readers have in regulating the biological activity of pRb.

Lamin B2 binding to minichromosome maintenance complex component 7 promotes non-small cell lung carcinogenesis.

We investigated the role of lamin B2 in non-small cell lung cancer (NSCLC). We detected higher lamin B2 expression in 20 NSCLC tumor tissues obtained from The Cancer Genome Atlas than in adjacent normal lung tissues. LMNB2-RNAi knockdown in A549 and H1299 NSCLC cells inhibited colony formation, cell proliferation and G1-S cell cycle progression while increasing apoptosis. LMNB2 overexpression had the opposite effects. Tumor xenograft experiments showed diminished tumor growth with LMNB2 knockdown H1299 cells than with controls. Yeast two-hybrid studies revealed minichromosome maintenance complex component 7 (MCM7) to be a binding partner of lamin B2, which was confirmed by co-immunoprecipitation and co-localization studies. Lamin B2 binding enhanced DNA binding and helicase activities of MCM7. Deletion analysis with MCM7-N, MCM7-M or MCM7-C mutant proteins showed that lamin B2 binds to the C-terminus of MCM7, and competes with the binding of the tumor suppressor retinoblastoma (RB) protein. Immunohistochemical analysis of 150 NSCLC patient samples revealed that both lamin B2 and MCM7 levels positively correlated with histological grade and tumor TNM stage. Moreover, high lamin B2 and MCM7 levels correlated with shorter overall survival of NSCLC patients. In sum, these results show that lamin B2 interaction with MCM7 promotes NSCLC progression.

ABT-263 induces G1/G0-phase arrest, apoptosis and autophagy in human esophageal cancer cells in vitro.

Both the anti- and pro-apoptotic members of the Bcl-2 family are regulated by a conserved Bcl-2 homology (BH3) domain. ABT-263 (Navitoclax), a novel BH3 mimetic and orally bioavailable Bcl-2 family inhibitor with high affinity for Bcl-xL, Bcl-2 and Bcl-w has entered clinical trials for cancer treatment. But the anticancer mechanisms of ABT-263 have not been fully elucidated. In this study we investigated the effects of ABT-263 on human esophageal cancer cells in vitro and to explore its anticancer mechanisms. Treatment with ABT-263 dose-dependently suppressed the viability of 3 human esophageal cancer cells with IC50 values of 10.7±1.4, 7.1±1.5 and 8.2±1.6 μmol/L, in EC109, HKESC-2 and CaES-17 cells, respectively. ABT-263 (5-20 μmol/L) dose-dependently induced G1/G0-phase arrest in the 3 cancer cell lines and induced apoptosis evidenced by increased the Annexin V-positive cell population and elevated levels of cleaved caspase 3, cleaved caspase 9 and PARP. We further demonstrated that ABT-263 treatment markedly increased the expression of p21Waf1/Cip1 and decreased the expression of cyclin D1 and phospho-Rb (retinoblastoma tumor suppressor protein) (Ser780) proteins that contributed to the G1/G0-phase arrest. Knockdown of p21Waf1/Cip1 attenuated ABT-263-induced G1/G0-phase arrest. Moreover, ABT-263 treatment enhanced pro-survival autophagy, shown as the increased LC3-II levels and decreased p62 levels, which counteracted its anticancer activity. Our results suggest that ABT-263 exerts cytostatic and cytotoxic effects on human esophageal cancer cells in vitro and enhances pro-survival autophagy, which counteracts its anticancer activity.

Abstract LB-087: Stage specific re-calibration of E2F1 function after RB loss

Abstract Prostate cancer (PCa) is the most frequently diagnosed non-cutaneous malignancy, and the third leading cause of cancer death in males in the United States. The development of the disease is reliant on the activity of the androgen receptor (AR), which promotes proliferation and is required for cell growth and survival. Deregulation of AR leads to the progression of PCa to the incurable form of disease termed castration-resistant prostate cancer (CRPC). One of the underlying mechanisms of AR deregulation is the loss of function of the retinoblastoma (RB) tumor suppressor protein. RB loss is almost exclusively seen in CRPC and is sufficient to induce a CRPC phenotype in primary PCa models. However, RB function is only lost in roughly 30% of tumors. The remaining 70% of tumors that progress to CRPC do so via other mechanisms and retain RB function. A subset of RB positive CRPC tumors progress to lose RB function even in this post-CRPC transition stage of disease. Biological assessment performed in isogenic RB knockdown, both hormone-sensitive and CRPC models, identified distinct functional consequences of RB loss depending on disease state. Transcriptome analysis identified opposing regulated gene signatures between disease stages. Further investigation of the E2F1 cistrome revealed differential E2F1 binding with RB loss in hormone sensitive and CRPC models, identifying distinct roles of E2F1, in the absence of RB, in different disease states. Data to be discussed reveal the stage-specific molecular pathways driven by E2F1 upon RB loss with the potential to promote disease progression. Citation Format: Amy C. Mandigo, Christopher McNair, Kexin Xu, Fugen Li, Matthew J. Schiewer, Myles Brown, Karen E. Knudsen. Stage specific re-calibration of E2F1 function after RB loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-087. doi:10.1158/1538-7445.AM2017-LB-087

A screen for inducers of bHLH activity identifies pitavastatin as a regulator of p21, Rb phosphorylation and E2F target gene expression in pancreatic cancer.

The average survival for patients with Pancreatic Ductal Adenocarcinoma (PDA) is merely 6 months, underscoring the need for new therapeutic approaches. During PDA progression, pancreatic acinar cells lose activity of the ClassI/II bHLH factors that regulate quiescence. We previously found that promoting transcriptional activity of the Class I bHLH factor E47 in highly aggressive PDA cells induced stable growth arrest in vitro and in vivo. To translate these findings for clinical utility, we developed a high throughput screening platform to identify small molecule inducers of Class I/II bHLH activity. A screen of 4,375 known drugs identified 70 bHLH activators. Prominent among the hits were members of the statin class of HMG-CoA reductase inhibitors, cholesterol lowering drugs that are also being evaluated in cancer. Studies with pitavastatin in primary patient derived tumor cells and established PDA lines, revealed dose dependent growth inhibition. At the molecular level, pitavastatin induced expression of the cyclin dependent kinase (CDK) inhibitor p21 in a cholesterol independent manner, blocked repressive phosphorylation of the Retinoblastoma tumor suppressor protein at CDK targeted sites, and reduced expression of E2F target genes required for progression through the G1/S boundary. Together, the data provide new insight into mechanisms by which statins constrain proliferation in cancer and establish the effectiveness of a novel screening platform to identify small molecules of clinical relevance in pancreatic cancer.

Cdc25A Is a Critical Mediator of Ischemic Neuronal Death In Vitro and In Vivo.

Dysregulation of cell cycle machinery is implicated in a number of neuronal death contexts, including stroke. Increasing evidence suggests that cyclin-dependent kinases (Cdks) are inappropriately activated in mature neurons under ischemic stress conditions. We previously demonstrated a functional role for the cyclin D1/Cdk4/pRb (retinoblastoma tumor suppressor protein) pathway in delayed neuronal death induced by ischemia. However, the molecular signals leading to cyclin D/Cdk4/pRb activation following ischemic insult are presently not clear. Here, we investigate the cell division cycle 25 (Cdc25) dual-specificity phosphatases as potential upstream regulators of ischemic neuronal death and Cdk4 activation. We show that a pharmacologic inhibitor of Cdc25 family members (A, B, and C) protects mouse primary neurons from hypoxia-induced delayed death. The major contributor to the death process appears to be Cdc25A. shRNA-mediated knockdown of Cdc25A protects neurons in a delayed model of hypoxia-induced death in vitro Similar results were observed in vivo following global ischemia in the rat. In contrast, neurons singly or doubly deficient for Cdc25B/C were not significantly protective. We show that Cdc25A activity, but not level, is upregulated in vitro following hypoxia and global ischemic insult in vivo Finally, we show that shRNA targeting Cdc25A blocks Ser795 pRb phosphorylation. Overall, our results indicate a role for Cdc25A in delayed neuronal death mediated by ischemia.SIGNIFICANCE STATEMENT A major challenge in stroke is finding an effective neuroprotective strategy to treat cerebral ischemic injury. Cdc25 family member A (Cdc25A) is a phosphatase normally activated during cell division in proliferating cells. We found that Cdc25A is activated in neurons undergoing ischemic stress mediated by hypoxia in vitro and global cerebral ischemia in rats in vivo We show that pharmacologic or genetic inhibition of Cdc25A activity protects neurons from delayed death in vitro and in vivo Downregulation of Cdc25A led to reduction in retinoblastoma tumor suppressor protein (pRb) phosphorylation. An increase in pRb phosphorylation has been previously linked to ischemic neuronal death. Our results identify Cdc25A as a potential target for neuroprotectant strategy for the treatment of delayed ischemic neuronal death.

A Mathematical Model of Cell Cycle Dysregulation Due to Human Papillomavirus Infection.

Human papillomaviruses (HPVs) that infect mucosal epithelium can be classified as high risk or low risk based on their propensity to cause lesions that can undergo malignant progression. HPVs produce the E7 protein that binds to cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein (RB) to modulate cell cycle control. Generally, high-risk HPV E7 proteins bind to RB with a higher affinity than low-risk HPV E7s, but both are able to deactivate RB and trigger S phase progression. In uninfected cells, RB inactivation is a tightly controlled process that must coincide with growth factor stimulation to commit cells to division. High-risk HPV E7 proteins short-circuit this control by decreasing growth factor requirement for cell division. We develop a mathematical model to examine the role that RB binding affinity, growth factor concentration, and E7 concentration have on cell cycle progression. Our model predicts that high RB binding affinity and E7 concentration accelerate the [Formula: see text] to S phase transition and weaken the dependence on growth factor. This model thus captures a key step in high-risk HPV oncogenesis.

Linker Histone H1.2 Directs Genome-wide Chromatin Association of the Retinoblastoma Tumor Suppressor Protein and Facilitates Its Function

SummaryThe retinoblastoma tumor suppressor protein pRb is a master regulator of cellular proliferation, principally through interaction with E2F and regulation of E2F target genes. Here, we describe the H1.2 linker histone as a major pRb interaction partner. We establish that H1.2 and pRb are found in a chromatin-bound complex on diverse E2F target genes. Interrogating the global influence of H1.2 on the genome-wide distribution of pRb indicated that the E2F target genes affected by H1.2 are functionally linked to cell-cycle control, consistent with the ability of H1.2 to hinder cell proliferation and the elevated levels of chromatin-bound H1-pRb complex, which occur in growth-arrested cells. Our results define a network of E2F target genes as susceptible to the regulatory influence of H1.2, where H1.2 augments global association of pRb with chromatin, enhances transcriptional repression by pRb, and facilitates pRb-dependent cell-cycle arrest.

Feasibility of cotargeting cell cycle and androgen signaling in metastatic hormone-sensitive prostate cancer (mHSPC).

151 Background: Most patients (pts) with mHSPC treated with androgen deprivation therapy (ADT) will progress to castration resistance. Androgens drive PC cell proliferation via upregulation of cyclin D which complexes with the cyclin-dependent protein kinases 4/6 (CDK4/6) resulting in phosphorylation of retinoblastoma (Rb) tumor suppressor protein and G1/S progression. Alterations in this pathway contribute to progression to castration-resistance (CR). In preclinical PC models, palbociclib, a novel specific CDK4/ 6 inhibitor, blocked proliferation and promoted G1 arrest in an Rb and Cyclin D dependent manner. Rb is lost in &lt;10% of localized PC, but &gt;30% of CR disease. Rb loss in mHSPC is estimated to be 10-20%. Based on this preclinical data, we designed a randomized phase II clinical trial (NCT02059213) to test the hypothesis that palbociclib + ADT in newly metastatic Rb-positive PC pts will significantly increase ADT efficacy. Methods: mHSPC pts with Rb intact tumors based on metastatic biopsy are stratified by disease extent and randomized (1:2) to ADT or ADT+ palbociclib. Primary endpoint is confirmed prostate specific antigen (PSA) response (≤ 4 ng/mL) after 28 weeks of therapy. With 20 patients randomized to ADT and 40 to ADT + palbociclib there will be a 64.2% power to detect a 20% difference in proportions with a one-sided type I error of 0.10 using the mid p-value method of the Fisher’s exact test. Secondary endpoints: safety and tolerability, rate of undetectable PSA (≤ 0.2ng/mL), biochemical and clinical PFS, overall PSA and radiographic RR, therapeutic biomarkers (circulating DNA and tumor cells, tumor protein and transcriptome) and to establish a repository of mHSPC samples. Results: Since July 2014, 68 patients have been registered and underwent tumor metastatic disease biopsy (36 soft tissue, 32 bone). 60/68 (88%) had adequate tissue for Rb assessment. 58/60 (97%) tumors were Rb positive (IHC). To date, 58/60 patients have been randomized to ADT (19 patients) or ADT + palbociclib (39 patients). Conclusions: Biopsy driven studies are feasible in the mHSPC population. Rb expression was present in 58/60 (97%) patients. This study is ongoing. Support: Movember-PCF Challenge Award, Pfizer. Clinical trial information: NCT02059213.

The Complex Link between Apoptosis and Autophagy: a Promising New Role for RB.

Physiological processes, as autophagy, proliferation and apoptosis are affected during carcinogenesis. Restoring cellular sensitivity to apoptotic stimuli, such as the antineoplastic cocktails, has been explored as a strategy to eliminate cancer cells. Autophagy, a physiological process of recycling organelles and macromolecules can be deviated from homeostasis to support cancer cells survival, proliferation, escape from apoptosis, and therapy resistance. The relationship between autophagy and apoptosis is complex and many stimuli can induce both processes. Most chemotherapeutic agents induce autophagy and it is not clear whether and how this chemotherapy-induced autophagy might contribute to resistance to apoptosis. Here, we review current strategies to sensitize cancer cells by interfering with autophagy. Moreover, we discuss a new link between autophagy and apoptosis: the tumor suppressor retinoblastoma protein (RB). Inactivation of RB is one of the earliest and more frequent hallmarks of cancer transformation, known to control cell cycle progression and apoptosis. Therefore, understanding RB functions in controlling cell fate is essential for an effective translation of RB status in cancer samples to the clinical outcome.

The antiviral cytokine interferon-gamma restricts neural stem/progenitor cell proliferation through activation of STAT1 and modulation of retinoblastoma protein phosphorylation.

Neural stem/progenitor cells (NPSCs) express receptors for many inflammatory cytokines, with varying effects on differentiation and proliferation depending on the stage of development and the milieu of inflammatory mediators. In primary neurons and astrocytes, we recently showed that interferon gamma (IFNγ), a potent antiviral cytokine that is required for the control and clearance of many central nervous system (CNS) infections, could differentially affect cell survival and cell cycle progression depending upon the cell type and the profile of activated intracellular signaling molecules. Here, we show that IFNγ inhibits proliferation of primary NSPCs through dephosphorylation of the tumor suppressor Retinoblastoma protein (pRb), which is dependent on activation of signal transducers and activators of transcription‐1 (STAT1) signaling pathways. Our results show i) IFNγ inhibits neurosphere growth and proliferation rate in a dose‐dependent manner; ii) IFNγ blocks cell cycle progression through a late‐stage G1/S phase restriction; iii) IFNγ induces phosphorylation and expression of STAT1 and STAT3; iv) IFNγ decreases cyclin E/cdk2 expression and reduces phosphorylation of cyclin D1 and pRb on serine residue 795; and v) the effects of IFNγ on NSPC proliferation, cell cycle protein expression, and pRb phosphorylation are STAT1‐dependent. These data define a mechanism by which IFNγ could contribute to a reduction in NSPC proliferation in inflammatory conditions. Further delineation of the effects of inflammatory cytokines on NSPC growth could improve our understanding of how CNS infections and other inflammatory events disrupt brain development and NSPC function. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Abstract B08: Retinoblastoma protein orchestrates cellular apoptosis in non-small cell lung cancer in response to CDK4/6 inhibition: novel targets and key mechanisms

Abstract Introduction: The retinoblastoma tumor suppressor protein (RB) is a critical mediator of cell proliferation via its regulation of cell cycle by binding to E2Fs. One of the mechanisms of RB activation is through CDK4/6 inhibition. Currently, multiple selective CDK4/6 inhibitors are being utilized in the clinical setting and are thought to drive cellular cytostasis. In this study, we demonstrate that RB activation not only results in cellular senescence, but for the first time, induces apoptosis by repressing IAPs (inhibitor of apoptosis protein). Experimental Procedures: Isogenic pairs of RB knockdown and control cells were interrogated in vitro, and in xenograft tumors for CDK 4/6 inhibitor response. Cellular senescence and apoptosis were quantified. Molecular mechanism was determined using microarray, chromatin immunoprecipitation, and co-immunoprecipitation assays. Summary: CDK4/6 inhibition diminished cell growth in an RB dependent manner both in in vitro and in vivo models. Immunohistochemistry from the xenografts demonstrated increased levels of apoptosis in the RB proficient isogenic lines. This was confirmed via western blot analysis for Caspase-3. Mechanistically, microarray analysis demonstrated that RB activation downregulated several key anti-apoptotic proteins (e.g. FOXM1, Survivin). ChIP assay and in silico modeling showed that FOXM1 and Survivin are repressed by RB/E2F binding. In the RB deficient setting, immunoprecipitation demonstrates that FOXM1 and Survivin interact directly with Caspase-3 resulting in decreased apoptosis. This was reversed using a competitive assay with overexpression of the SMAC (second mitochondrial) protein. Conclusion: Data from multiple isogenic NSCLC models demonstrate that RB activation via CDK4/6 inhibitors results in apoptosis in an RB dependent manner. Mechanistically, RB represses transcription of multiple IAPs (including FOXM1 and Survivin), which allows pro-apoptotic factors such as SMAC to activate Caspase-3. These findings suggest that CDK4/6 inhibitors should be viewed as cytotoxic, as opposed to purely cytostatic, agents and warrant further clinical investigation. Citation Format: Chellappagounder Thangavel, Ettickan Boopathi, Yi Liu, Alex Haber, Maryna Perepelyuk, Sankar Addya, Sunday Shoyele, Adam P. Dicker, Karen E. Knudsen, Robert B. Den. Retinoblastoma protein orchestrates cellular apoptosis in non-small cell lung cancer in response to CDK4/6 inhibition: novel targets and key mechanisms. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B08.

Abstract IA06: Co-Targeting cell cycle and androgen signaling to personalize therapy for hormone dependent prostate cancer

Abstract Prostate cancer (PC) is an androgen driven and dependent disease and androgen deprivation therapy (ADT) is the standard for patients with metastatic hormone sensitive disease. Despite a high response rate, most patients will progress to castration resistance. The expanding molecular knowledge highlights the biologic heterogeneity and adaptive capacity of PC, and provides the rationale for a preemptive multi-targeted therapeutic strategy. Compelling support for the latter strategy is provided by recent data from two randomized trials demonstrating an unprecedented impact on survival with the addition of docetaxel to ADT. Androgens drive proliferation of PC cells via upregulation of cyclin D which complexes with the cyclin-dependent protein kinases 4/6 (CDK4/6) resulting in phosphorylation of retinoblastoma (Rb) tumor suppressor protein and G1/S progression. Alterations in this pathway contribute to progression to castration-resistance. Palbociclib is a novel specific CDK4/ 6 inhibitor which was recently approved in combination with letrozole for postmenopausal women with ER +, HER2 negative advanced breast cancer. In preclinical prostate cancer models, palbociclib inhibited proliferation and promoted G1 arrest in an Rb and Cyclin D dependent manner. Current data indicate that wild-type Rb expression is present in in 95% vs 75% of patients with newly diagnosed vs metastatic castration resistant PC respectively. We estimate that 80-90% of early metastatic hormone-sensitive PC will harbor intact Rb. Based on the biologic and preclinical data, we developed a multicenter randomized phase II clinical trial (NCT02059213) to test the hypothesis that adding palbociclib to ADT in patients with newly metastatic Rb-positive PC will significantly increase the efficacy of ADT. Patients (n = 60) with new metastatic hormone sensitive PC and Rb intact tumors based on metastatic biopsy are stratified by disease extent and randomized (1:2) to ADT or ADT+ palbociclib. Primary endpoint is confirmed prostate specific antigen (PSA) response (&amp;lt; 4 ng/mL) after 28 weeks of therapy. With 20 patients randomized to ADT and 40 randomized to ADT plus palbociclib there will be a 64.2% power to detect a 20% difference in proportions with a one-sided type I error of 0.10 using the mid p-value method of the Fisher's exact test. Secondary endpoints: safety and tolerability of ADT + palbociclib, rate of undetectable PSA (&amp;lt; 0.2ng/mL), biochemical and clinical progression-free survival, overall PSA and radiographic response rates, assessment of biomarkers which predict therapy response (circulating DNA and tumor cells, tumor protein and transcriptome analysis) and to establish a repository of metastatic hormone sensitive prostate tumor samples. To date 39 patients have been registered and underwent tumor metastatic disease biopsy (soft tissue 26, bone 13). 2 patients were Rb negative and 5 had inadequate tissue. 32 patients have been randomized to ADT (11 patients) or ADT + palbociclib (21 patients). The study is in progress. Support: Movember-PCF Challenge Award, Pfizer. Citation Format: Maha Hussain, Phillip L. Palmbos1, Scott A. Tomlins1, Stephanie Daignault-Newton, Neeraj Agarwal, Przemyslaw Twardowski, Alicia K. Morgans, Emmanuel S. Antonarakis, Karen E. Knudsen, Felix Y. Feng. Co-Targeting cell cycle and androgen signaling to personalize therapy for hormone dependent prostate cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA06.

Abstract B11: Re-wired E2F function in response to RB loss as a potential driver of castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is the most frequently diagnosed non-cutaneous malignancy, and second leading cause of cancer death in males in the United States, causing over 30,000 deaths per year. A crucial component to the development and progression of PCa is the activity of the androgen receptor (AR). AR promotes proliferation and is required for PCa cell growth and survival. As such, targeting the AR-signaling axis through androgen deprivation therapy (ADT) is the first line of therapy for PCa. However, cells invariably become resistant to this therapy and men relapse with the incurable form of the disease, castration-resistant prostate cancer (CRPC). It has been shown that an underlying mechanism of this resistance revolves around the retinoblastoma (RB) tumor suppressor protein. RB functions to repress tumor development by negatively regulating the activity of the E2F family of transcription factors thus, resulting in a decrease in AR levels. RB additionally controls cell cycle progression through its ability to bind and inhibit E2F1 mediated transcription of genes required for G1/S progression. Surprisingly, genome-wide assessment of E2F activity in the absence of RB has been understudied. To better understand the molecular pathways that are altered upon RB loss and the roles they play in CRPC, RNA-Seq and ChIP-Seq analyses were performed in isogenic RB cell lines in both hormone sensitive and CRPC models. Data to be discussed reveal new insight into the differential effects of RB loss on AR and E2F1 function and role as potential drivers of late stage disease. Citation Format: Amy C. Mandigo, Chris McNair, Matthew J. Schiewer, Kexin Xu, Fugen Li, Brown Myles, Karen E. Knudsen. Re-wired E2F function in response to RB loss as a potential driver of castration-resistant prostate cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B11.

Abstract IA15: The consequences of pRb inactivation: insights from a proteomic analysis of Rb loss

Abstract The retinoblastoma tumor suppressor protein associates with chromatin and regulates gene expression. The transcriptional signatures associated with RB1/Rb mutation are thought to give a picture of the cellular changes that occur when pRB is lost. We used proteomic profiling to examine the changes caused by the ablation of Rb in mouse lung and colon tissues, and compared these with transcript profiles. While the transcription of classic E2F target genes increased similarly in Rbko lung and colon, effects on protein levels were context-dependent. Proteomic changes were identified that were similar between Rb-mutant tissues but, unexpectedly, the major feature of these changes was a decrease in proteins that function in mitochondria. Consistent with this, mutation of RB1 in cultured human cells reduced the number of mitochondrial number and caused mitochondrial dysfunction. Rbko cells had reduced oxygen consumption rate, reduced reserve capacity, an accumulation of depolarized mitochondria and displayed an altered flux of Carbon through the TCA cycle that was evident both in vivo and in vitro. These defects impair cell proliferation under conditions of mitochondria stress. Collectively, these results suggest that the most consistent changes in the proteome of Rbko tissues may not stem from changes in the levels of cell proliferation proteins, but from changes in mitochondrial function. Analysis of transcripts that are induced in Rbko tissues without a corresponding change in protein levels suggests that there may be a mechanism of post-transcriptional control that coordinately suppresses E2F-induced mRNAs. Citation Format: Brandon N. Nicolay, Paul S. Danielian, Filippos Kottakis, John D. Lapek, Jr., Ioannis Sanidas, Wayne O. Miles, Mantre Dehnad, Katrin Tschöp, Jessica J. Gierut, Amity L. Manning, Robert Morris, Kevin Haigis, Nabeel Bardeesy, Jacqueline A. Lees, Wilhelm Haas, Nicholas J. Dyson. The consequences of pRb inactivation: insights from a proteomic analysis of Rb loss. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA15.

Dephosphorylation of the Retinoblastoma protein (Rb) inhibits cancer cell EMT via Zeb

ABSTRACTThe tumor suppressor Retinoblastoma (Rb) protein is highly phosphorylated in cancer cells largely due to the overexpression of cyclins or the loss of expression of cyclin dependent kinase inhibitors (cdki). Hyperphosphorylation of Rb promotes proliferation, and plays a role in the regulation of apoptosis. Recently, inhibition of cyclin dependent activity toward Rb has been identified as a strategy that has shown clinical efficacy. We utilized a method to induce phosphatase activity toward Rb in cells by shRNA silencing of PNUTS (Phosphatase Nuclear Targeting Subunit) that regulates PP1-mediated dephosphorylation of Rb. In this study, the effect of Rb dephosphorylation on the epithelial to mesenchymal transition (EMT) was determined. The EMT transition is observed in cancer cells that have acquired invasive characteristics. In breast cancer cells grown in 3D Matrigel cultures, MCF7 cells undergo apoptosis in response to Rb dephosphorylation, whereas MDA-MB-231 and Hs578T cells exhibit a reduction in the EMT. Cells devoid of phosphorylated Rb (nontransformed MCF10A and Rb-null MDA-MB-468) lacked any response to PNUTS depletion, showing the effect is Rb-dependent. In addition, these studies showed that Rb dephosphorylation in 3D Matrigel cultures of highly invasive HT1080 cells led to the inhibition of the EMT. Furthermore we observed association between dephosphorylated Rb with ZEB1, a zinc-finger E-box-binding transcription factor that regulates expression of E- and N-cadherins. Finally Rb dephosphorylation led to inhibition of ZEB1 transcriptional activity, this data supports the notion that Rb dephosphorylation modulates the EMT. These studies suggest targeting Rb phosphorylation in mesenchymal cancer cells may decrease invasiveness.

APC/C and retinoblastoma interaction: cross-talk of retinoblastoma protein with the ubiquitin proteasome pathway.

The ubiquitin (Ub) ligase anaphase promoting complex/cyclosome (APC/C) and the tumour suppressor retinoblastoma protein (pRB) play key roles in cell cycle regulation. APC/C is a critical regulator of mitosis and G1-phase of the cell cycle whereas pRB keeps a check on proliferation by inhibiting transition to the S-phase. APC/C and pRB interact with each other via the co-activator of APC/C, FZR1, providing an alternative pathway of regulation of G1 to S transition by pRB using a post-translational mechanism. Both pRB and FZR1 have complex roles and are implicated not only in regulation of cell proliferation but also in differentiation, quiescence, apoptosis, maintenance of chromosomal integrity and metabolism. Both are also targeted by transforming viruses. We discuss recent advances in our understanding of the involvement of APC/C and pRB in cell cycle based decisions and how these insights will be useful for development of anti-cancer and anti-viral drugs.

Histopathological transformation to small-cell lung carcinoma in non-small cell lung carcinoma tumors.

Lung cancer is the principal cause of cancer-related death worldwide. The use of targeted therapies, especially tyrosine kinase inhibitors (TKIs), in specific groups of patients has dramatically improved the prognosis of this disease, although inevitably some patients will develop resistance to these drugs during active treatment. The most common cancer-associated acquired mutation is the epidermal growth factor receptor (EGFR) Thr790Met (T790M) mutation. During active treatment with targeted therapies, histopathological transformation to small-cell lung carcinoma (SCLC) can occur in 3-15% of patients with non-small-cell lung carcinoma (NSCLC) tumors. By definition, SCLC is a high-grade tumor with specific histological and genetic characteristics. In the majority of cases, a good-quality hematoxylin and eosin (H&E) stain is enough to establish a diagnosis. Immunohistochemistry (IHC) is used to confirm the diagnosis and exclude other neoplasia such as sarcomatoid carcinomas, large-cell carcinoma, basaloid squamous-cell carcinoma, chronic inflammation, malignant melanoma, metastatic carcinoma, sarcoma, and lymphoma. A loss of the tumor-suppressor protein retinoblastoma 1 (RB1) is found in 100% of human SCLC tumors; therefore, it has an essential role in tumorigenesis and tumor development. Other genetic pathways probably involved in the histopathological transformation include neurogenic locus notch homolog (NOTCH) and achaete-scute homolog 1 (ASCL1). Histological transformation to SCLC can be suspected in NSCLC patients who clinically deteriorate during active treatment. Biopsy of any new lesion in this clinical setting is highly recommended to rule out a SCLC transformation. New studies are trying to assess this histological transformation by noninvasive measures such as measuring the concentration of serum neuron-specific enolase.