pRb Status Research Articles

Abstract PR011: Downregulation of the HIF2a target cyclin D1 underlies the efficacy of the HIF2a inhibitor belzutifan in kidney cancer

Abstract Abstract: Most clear cell renal cell carcinomas (ccRCCs) are caused by loss of the pVHL tumor suppressor function. Inactivation of pVHL leads to increased accumulation of HIF2a, which drives ccRCC in preclinical models. HIF2a interacts with ARNT and functions as a heterodimeric transcription factor. HIF2a can activate hundreds of downstream target genes, including many that are suspected of playing a role in ccRCC pathogenesis based on their biological functions. Peloton Therapeutics developed a specific allosteric HIF2a inhibitor named PT2399, which disrupts the HIF2a-ARNT interaction and silences the HIF2a transcription program. Furthermore, PT2399 suppresses the growth of ccRCC cells in in-vitro and in-vivo assays. A more advanced version of PT2399, Belzutifan, was recently approved for treating tumors in patients with germline VHL mutations (VHL disease) and is in Phase 3 testing for sporadic ccRCCs. Since HIF2a is a transcriptional activator, we hypothesized that PT2399’s ability to suppress ccRCC is linked to repression of one or more HIF2a target genes. To identify these genes, we performed a pooled whole genome CRISPR activation (CRISPRa) screen in the presence or absence of PT2399. This screen identified multiple genes (“hits”) that, when activated, likely conferred resistance to PT2399. To further differentiate between direct vs indirect HIF2a targets that confer resistance to PT2399, we performed nascent RNA-seq (Pro-seq) after acute treatment of ccRCC cells with PT2399. Comparison of the CRISPRa and Pro-Seq data, together with secondary validation assays, identified CCND1, which encodes Cyclin D1, as a direct target gene of HIF2a that, when activated, confers complete resistance to PT2399 in multiple ccRCC cell lines. Additionally, we showed that wild-type Cyclin D1 (WT), but not a mutant (KE) that fails to activate CDK4/6, confers resistance to PT2399. Interestingly, Cyclin D1 overrides the growth inhibitory effect of PT2399 in ccRCC cells irrespective of pRB status. To identify the relevant Rb-independent substrates of Cyclin D1, we performed IP-MS experiments in RB1-/- ccRCC cells under substrate trapping conditions. Our preliminary data from these experiments suggests that RBL1 (p107) and RBL2 (p130) substitute for pRB in RB1-/- ccRCC cells. Our findings provide further support for CDK4/6 as a therapeutic target in ccRCC. Citation Format: Nitin H. Shirole, Devishi Kesar, William R. Sellers, William G. Kaelin Jr. Downregulation of the HIF2a target cyclin D1 underlies the efficacy of the HIF2a inhibitor belzutifan in kidney cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR011.

The role of E2F1-topoIIβ signaling in regulation of cell cycle exit and neuronal differentiation of human SH-SY5Y cells

This study aims to test the role of E2F1-topoIIβ signaling in neuronal differentiation of SH-SY5Y cells. With retinoic acid (RA) induction, a high percentage of cells were found to be arrested at the G0/G1 phase, with decreased levels of cyclinD1, CDK4, phosphorylation status of pRb and E2F1, in addition to an elevated level of p27. The cells were shown to differentiate into neuronal phenotypes characterized by highly expressed neuronal markers, MAP2 and enriched topoIIβ, and remarkable neurite outgrowth. Exogenously forced E2F1 expression with a specific E2F1 plasmid led to suppression of topoIIβ expression and disruption of the neuronal differentiation of SH-SY5Y cells. On further examination using the ChIP assay, we found that E2F1 bound directly to the promoter region of topoIIβ, and its binding ability was inversely correlated with topoIIβ expression in response to RA induction. Thus, our findings suggest that E2F1-topoIIβ signaling may play a role in regulation of cell cycle exit and neuronal differentiation.

Abstract 4201: Combination of palbociclib with enzalutamide shows in vitro activity in RB-proficient and androgen receptor-positive triple-negative breast cancer cells

Abstract Background Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors have in vitro synergistic effects and significant clinical efficacy in combination with hormone therapy for estrogen receptor (ER)-positive advanced breast cancer. However, the effects of CDK4/6 inhibitors in triple negative breast cancer (TNBC) are not well-elucidated. Retinoblastoma (RB, a known substrate of CDK4/6) protein pathway deregulation is a frequent occurrence in TNBC and in vitro studies have revealed that pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin in RB-proficient TNBC models, suggesting that RB may be a biomarker for selecting patients receiving CDK4/6 inhibitors. In addition, anti-androgen therapy have been shown preclinical efficacy in androgen-receptor (AR) positive TNBC cells. Here we tested the combination effect of a CDK4/6 inhibitor palbociclib with an AR antagonist enzalutamide in TNBC cells. Methods MDA-MB-231, MDA-MB-468 and MDA-MB-453 TNBC cell lines were used for in vitro studies. Cell viability was examined by MTT assay. Apoptotic effects were examined by flow cytometry. Signal transduction pathways in cells were assessed by Western blot. Results We correlated the expressions of AR and pRB status in these TNBC cells and identified that MDA-MB-453 were AR-positive/pRB-positive, MDA-MB-231 were AR-negative/pRB-positive, and MDA-MB-468 were AR-negative/pRB-negative cells. Since CDK4/6 inhibition leads to dephosphorylation of pRB, we examined effects of palbociclib and found that palbociclib significantly inhibited the cell growth of pRB-positive cell lines MDA-MB-453 and MDA-MB-231, but did not affect pRB-negative MDA-MB-468 cells. Further combining palbociclib with enzalutamide showed that the growth inhibitory effects of combination therapy were additive in AR-positive/pRB-positive MDA-MB-453 cells, but not in AR-negative/pRB-positive MDA-MB-231 cells, nor AR/pRB double negative MDA-MB-468 cells. Moreover, palbociclib-induced G1/S arrest only exhibited in pRB-proficient MDA-MB-453 and MDA-MB-231 cells. In contrast, enzalutamide did not affect the cell cycle on any of these TNBC cell lines but inhibited phosphorylation of AR signaling in AR-positive MDA-MB-453 cells. In addition, both palbociclib and enzalutamide induced little apoptosis, suggesting cytostatic effects of these agents. Conclusions In summary, both pRB and AR status are important for response of TNBC cells to palbociclib. Combination of palbociclib with enzalutamide shows more prominent cytostatic effects in RB proficient and AR positive TNBC cells, comparing to AR-negative/pRB-positive or AR/pRB double negative TNBC cells. Our data provide a preclinical rationale in selecting patients who might have therapeutic benefit from combining CDK4/6 inhibitors with AR antagonists. Citation Format: Ling-Ming Tseng, Tzu-Ting Huang, Chun-Teng Huang, Ka-Yi Lau, Chun-Yu Liu, Chung-Wai Shiau, Kuen-Feng Chen. Combination of palbociclib with enzalutamide shows in vitro activity in RB-proficient and androgen receptor-positive triple-negative breast cancer cells [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 4201. doi:10.1158/1538-7445.AM2017-4201

Phosphorylation of pRb: mechanism for RB pathway inactivation in MYCN-amplified retinoblastoma.

A small, but unique subgroup of retinoblastoma has been identified with no detectable mutation in the retinoblastoma gene (RB1) and with high levels of MYCN gene amplification. This manuscript investigated alternate pathways of inactivating pRb, the encoded protein in these tumors. We analyzed the mutation status of the RB1 gene and MYCN copy number in a series of 245 unilateral retinoblastomas, and the phosphorylation status of pRb in a subset of five tumors using immunohistochemistry. There were 203 tumors with two mutations in RB1 (RB1 −/−, 83%), 29 with one (RB1 +/−, 12%) and 13 with no detectable mutations (RB1 +/+, 5%). Eighteen tumors carried MYCN amplification between 29 and 110 copies: 12 had two (RB1 −/−) or one RB1 (RB1 +/−) mutations, while six had no mutations (RB1 +/+). Immunohistochemical staining of tumor sections with antibodies against pRb and phosphorylated Rb (ppRb) displayed high levels of pRb and ppRb in both RB1 +/+ and RB1 +/− tumors with MYCN amplification compared to no expression of these proteins in a classic RB1 −/−, MYCN‐low tumor. These results establish that high MYCN amplification can be present in retinoblastoma with or without coding sequence mutations in the RB1 gene. The functional state of pRb is inferred to be inactive due to phosphorylation of pRb in the MYCN‐amplified retinoblastoma without coding sequence mutations. This makes inactivation of RB1 by gene mutation or its protein product, pRb, by protein phosphorylation, a necessary condition for initiating retinoblastoma tumorigenesis, independent of MYCN amplification.

Loss of the retinoblastoma tumor suppressor correlates with improved outcome in patients with lung adenocarcinoma treated with surgery and chemotherapy

The retinoblastoma tumor suppressor pathway is frequently inactivated in human cancer, enabling unrestrained proliferation. Most cancers, however, maintain expression of a wild-type (WT) retinoblastoma tumor suppressor protein (pRB). It is generally in a hyperphosphorylated state (ppRB) because of mutations in upstream regulators such as p16 and cyclin D. Hyperphosphorylated ppRB is considered inactive, although data are emerging that suggest it can retain some function. To test the clinical relevance of pRB status, we obtained archival tissue sections from 91 cases of lung adenocarcinoma resected between 2003 and 2008. All cases received platinum doublet chemotherapy, and the median survival was 5.9 years. All cases were assessed for pRB and ppRB using immunohistochemistry and quantified based on intensity of signal and proportion of positive cells. pRB expression was lost in 15% of lung adenocarcinoma cases. In tumors that did not express pRB, the survival rate was significantly improved (hazard ratio, 0.21; 95% confidence interval, 0.06-0.69; P = .01) in comparison to tumors that express pRB. pRB status was found to be an independent predictor of overall survival on multivariate analysis (hazard ratio, 0.22; 95% confidence interval, 0.07-0.73; P = .01) along with increased stage and age. pRB status did not alter baseline levels of apoptotic or proliferative markers in these tumors, but the DNA damage response protein 53BP1 was higher in cancers with high levels of pRB. In summary, loss of pRB expression is associated with improved survival in patients treated with surgical resection and chemotherapy. This may be useful in classifying patients at greatest benefit for aggressive treatment regimes.

Abstract 5155: Status and clinical relavence of G1/S cell cycle regulatory proteins in Ocular Surface Squamous Neoplasia

Abstract Purpose: Ocular Surface Squamous Neoplasia (OSSN) is a common tumour of ocular surface arising from the limbus, cornea or conjunctiva. It includes conjunctival intraepithelial neoplasia (CIN) and squamous cell carcinoma (SCC). Although confined to ocular surface, they have capability to recur (5-53%), metastasize (0-16%) and even cause death (8%).One of the most important issues still unresolved in the treatment and management of OSSN is the inability to determine factors causing disease progression. Identification of reliable biomarkers which could detect progression as well as identify high risk cases is an urgent necessity. The Cyclin D1/p16INK4a/pRb pathway plays a critical role in tumorigenesis and alterations in these proteins have been reported in various malignancies. This study was designed to explore the status of pRb, p16INK4a and cyclin D1 in various clinical stages of OSSN patients. Methods:Sixty-four histopathologically confirmed OSSN cases (44 squamous cell carcinomas and 20 conjunctival intraepithelial neoplasias) were included in this study. AJCC TNM staging was done and patients were followed up for 36 to 58 months (median 46 months). Immunohistochemical expression of total pRb (clone-13A10), phosphorylated Rb (clone-Phospho-Ser780), p16INK4a (clone-6H12), and cyclin D1 (clone-SP4) protein was evaluated. Kaplan-Meier survival and Cox regression analysis was done to assess the prognostic significance of these proteins. Results: Immunoexpression of the targeted proteins demonstrated loss /inactive form of pRb in 87% OSSN cases, p16INK4a and cyclin D1 overexpression in 28% in 63% cases respectively. From T1 to T3 AJCC category there was a progressive increase of p16INK4a (0%in T1 to 100% in T3) (P = 0.007) and cyclin D1 overexpression (25% in T1 to 88% in T4). Loss of pRb (75% in T1 to 100% in T4) (P = 0.03) was also seen. Loss of pRb expression was the earliest change which was detected in 75% cases in T1.The Kaplan Meier survival curves revealed loss of pRb (P = 0.0002, log rank analysis) as well as p16INK4a(P = 0.11, log rank analysis) and cyclin D1 over expression (P = 0.06, log rank analysis) adversely affected the disease free survival. pRb loss however emerged as the single most sensitive poor prognostic indicator in all AJCC stages of OSSN patients. Conclusion: G1/S cell cycle proteins (retinoblastoma, p16INK4aand cyclin D1) have prognostic relevance in OSSN patients. Of these pRb loss is the most useful indicator of aggressive behavior and is therefore recommended for detecting high risk OSSN patients. The dysregulation of p16INK4a, cyclin D1 and pRb proteins also plays an important role in pathogenesis of OSSN patients. Note: This abstract was not presented at the meeting. Citation Format: Sheetal Chauhan, Seema Sen, Anjana Sharma, Seema Kashyap, M. Vanathi, Neelam Pushker, Radhika Tandon. Status and clinical relavence of G1/S cell cycle regulatory proteins in Ocular Surface Squamous Neoplasia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5155. doi:10.1158/1538-7445.AM2015-5155

Abstract 5567: Impact of progesterone receptor (PR) isotype-specific immunohistochemistry (IHC) on the diagnosis of triple negative breast cancer (BC)

Abstract Background: Given the poor therapeutic outcomes for triple negative BC, diagnostic accuracy is vital. In routine IHC, PR is measured using bispecific antibodies (Ab) that recognize epitopes common to PRA and PRB. PR A and B expression is imbalanced in BC. Relative expression of the PR isotypes appears to be prognostic in tumors evaluated with a bispecific Ab (Hopp 2004). Tumors can express PRA or PRB on different cells in the same tumor (Mote 2008), which differ from those expressing ERα (Zukiwski 2013). A true triple negative phenotype might escape detection with the use of one single Ab to detect both PR A and B epitopes, depending on its sensitivity/specificity. This study evaluated the use of two isotype-specific PR Abs to fully characterize PR status. Methods: 312 archived BC specimens with clinical data came from Oscar Lambret Cancer Center, Lille. HER2 status was previously determined. IHC was performed using anti-ERα, anti-PRA and PRB Abs. ERα and PR tumor positivity was defined as ≥ 1% stained tumor cells. Results: 276 cases had complete data. Table 1 shows ERα, PRA and PRB status. 38/276 cases (14%) were ERα and HER2 negative: 22 were PR A or B negative, 9 were PR A and B positive, 2 were positive only for PRA and 5 only for PRB. Discussion: Bispecific PR Abs rarely report PRA and PRB similarly (Mote 2001). PR Phosphorylation and activation may vary across tumors. Potentially only one PR isotype may drive BC tumor growth. Triple-negative BC status determination could be PR Ab-dependent. These cases are currently being tested with a standard bispecific PR Ab to understand which would be considered triple-negative with routine IHC. Conclusion: Further evaluation of IHC with specific PRA and PRB Abs is warranted to determine if certain patients, currently classified as having triple-negative BC, could respond to antiprogestins, which have shown activity in BC. Table 1% of276 casesPR (PRA or PRB)PRAPRBERαNegativePositiveTotalNegativePositiveTotalNegativePositiveTotalNegative148221742115722Positive77178146579106868Total217910031691002575100 Citation Format: Erard Gilles, Jacques Bosq, Laura Caplier, Alice Bexon, Alexander Zukiwski, Jacques Bonneterre. Impact of progesterone receptor (PR) isotype-specific immunohistochemistry (IHC) on the diagnosis of triple negative breast cancer (BC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5567. doi:10.1158/1538-7445.AM2014-5567

E2F-1 binding affinity for pRb is not the only determinant of the E2F-1 activity

E2F-1 is the major cellular target of pRB and is regulated by pRB during cell proliferation. Interaction between pRB and E2F-1 is dependent on the phosphorylation status of pRB. Despite the fact that E2F-1 and pRB have antagonistic activities when they are overexpressed, the role of the E2F-1-pRB interaction in cell growth largely remains unknown. Ideally, it would be better to study the properties of a pRB mutant that fails to bind to E2F, but retains all other activities. To date, no pRB mutation has been characterized in sufficient detail to show that it specifically eliminates E2F binding but leaves other interactions intact. An alternative approach to this issue is to ask whether mutations that change E2F proteins binding affinity to pRB are sufficient to change cell growth in aspect of cell cycle and tumor formation. Therefore, we used the E2F-1 mutants including E2F-1/S332-7A, E2F-1/S375A, E2F-1/S403A, E2F-1/Y411A and E2F-1/L132Q that have different binding affinities for pRB to better understand the roles of the E2F-1 phosphorylation and E2F-1-pRB interaction in the cell cycle, as well as in transformation and gene expression. Data presented in this study suggests that in vivo phosphorylation at amino acids 332-337, 375 and 403 is important for the E2F-1 and pRB interaction in vivo. However, although E2F-1 mutants 332-7, 375 and 403 showed similar binding affinity to pRB, they showed different characteristics in transformation efficiency, G(0) accumulation, and target gene experiments.

Abstract C191: The effect of the cyclin-dependent kinase inhibitor PD 0332991 on glioblastoma cells and relationship with p15INK4B/p16INK4A expression

Abstract Glioblastoma multiforme (GBM) is the most common and highly malignant primary brain tumor in adults with a median survival just over 1 year, and novel therapies are urgently needed. Deregulation of the p16 (INK4A)-CDK4/6-pRb pathway are commonly found in human GBM patients and selective inhibitors of CDK4/6 may be specifically useful to suppress proliferation associated with these alterations. PD0332991 is an orally active small molecule that specifically inhibits CDK4 and CDK6. Therefore, the efficacy of PD0332991 was evaluated in the Mayo panel of primary GBM xenograft lines using short-term explant cultures. We hypothesized that expression of p15 and/or p16, endogenous inhibitors of CDK4/6 activity, would suppress the response of GBM tumors to PD0332991 treatment. To test this hypothesis, 2 GBM xenograft cell lines with wild-type p16/p15 levels (GBM5 and 28) and 2 lines without detectable p16/p15 levels (GBM22 and 43) were selected for further analysis. Tumor cells were treated with graded concentrations (10 nanoM to1 microM) of PD0332991 for 5 days and subjected to CyQUANT cell proliferation assays. Consistent with our hypothesis, treatment with 1 microM PD0332991 effectively inhibited cell proliferation to 56.6±3.5% (P<0.001) and 44.3±3.4% (P<0.001) of control in GBM22 and GBM43 cells respectively, but did not affect cell proliferation in GBM5 and GBM28 cells (98.7±15.6% and 101.8±12.0% of control, respectively). In addition, 1 microM PD0332991 induced G1-phase cell cycle arrest in GBM22 (treated 79.7±0.8% vs. control 63.4±0.5% of cells in G1 phase, P<0.001) and GBM43 (treated 70.6±1.1% vs. control 48.9±4.6% of cells in G1 phase, P<0.001) but not in GBM5 and GBM28 (treated 52.3±2.7% vs. control 54.0±5.1%, treated 52.4±4.7% vs. control 54.6±2.6%, respectively). The status of p15 and p16 was confirmed by RT-PCR, which demonstrated readily detectable expression of p15 and p16 in GBM5 and GBM28, but not in GBM22 and GBM43 cells. Additional comparative genomic hybridization (CGH) data showed that CDKN2A and 2B (genes encode for p16 and p15 respectively) are homozygously deleted in GBM22 and 43, hemizygously deleted in GBM5 and normal in GBM28. We also examined the status of CDK4, CDK6, pRb and other molecules in the p16-CDK4/6-pRb pathway by Western, RT-PCR and CGH in these GBM lines and the data will be presented. We are now testing whether shRNA knockdown and/or re-expression of p15/p16 in the relevant cell lines alters their responsiveness to PD0332991. In conclusion, CDK4/6 inhibitor PD0332991 inhibits cell proliferation and induces G1 cell cycle arrest in p15/p16- null GBM tumors, but is less effective in p15/p16 expressing tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C191.

The use of an immunohistochemical diagnostic panel to determine the primary site of cervical lymph node metastases of occult squamous cell carcinoma

Cervical lymph node metastases from unknown primary sites account for approximately 3% to 9% of all head and neck malignant lesions. Squamous cell carcinoma is the most common type of cervical metastatic carcinoma. Our aim was to investigate the possibility of determining the site of primary tumors using an immunohistochemical diagnostic panel in metastatic cervical lymph nodes. Expression profiles of cytokeratins, 5/6; 8/18; 10; 13; 14; and 19, p16, and pRb were evaluated in 101 consecutive patients with cervical nodal metastasis who had undergone neck dissection to treat known head and neck squamous cell carcinoma (primary sites: 16, oral cavity; 38, oropharynx; 26, hypopharynx; 21, larynx). Cytokeratin 10 was more frequently expressed in oral cavity primary tumors, whereas cytokeratin 19 staining was more frequently observed in tumors originated from the pharynx and larynx. The expression of p16 and altered pRb status (0% or >50%) were more frequently observed in oropharynx primary tumors. To select the best subset among the 8 antibodies tested, classification and regression tree analysis was performed. The analysis correctly classified the four primary sites (25.0% of oral cavity, 89.5% of oropharynx, 30.8% of hypopharynx, and 57.1% of larynx) using 5 variables (histologic subtype, p16, cytokeratins 10 and 19, and pRb). The p16 was the single best predictor. The classification tree method using immunostaining profiles of p16, cytokeratins 10 and 19, or pRb may be helpful in the identification of the primary site of metastatic squamous cell carcinoma with occult primary.

High prevalence of retinoblastoma protein loss in triple-negative breast cancers and its association with a good prognosis in patients treated with adjuvant chemotherapy

BackgroundTriple-negative breast cancer (TNBC) is an aggressive disease, nevertheless exhibiting a high response rate to chemotherapy. Since the retinoblastoma protein (pRb) loss confers a high sensitivity to chemotherapy regimens, we evaluated the prevalence of pRb loss in TNBCs and its relevance on the clinical outcome of patients treated with adjuvant chemotherapy. Patients and methodspRb status was prospectively evaluated by immunocytochemistry in 518 consecutive patients with complete receptor information. The predictive value of pRb status in TNBCs was determined according to the adjuvant therapeutic treatments. ResultsFifty-three tumors were identified as TNBCs. The prevalence of pRb loss was significantly higher in TNBCs than in the other cancer subtypes. All patients with TNBCs lacking pRb and treated with systemic chemotherapy (cyclophosphamide, methotrexate and 5-fluorouracil) were disease free at a medium follow-up time of 109 months, whereas the clinical outcome of those expressing pRb was significantly poorer (P = 0.008). Analysis of disease-free survival including the established anatomo-clinical prognostic parameters indicated pRb loss as the only significant predictive factor. ConclusionspRb loss is much more frequent in TNBCs than in the other breast cancer subtypes. Patients with TNBCs lacking pRb had a very favorable clinical outcome if treated with conventional adjuvant chemotherapy.

Runx2, p53, and pRB status as diagnostic parameters for deregulation of osteoblast growth and differentiation in a new pre‐chemotherapeutic osteosarcoma cell line (OS1)

Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS-2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre-chemotherapeutic tumor in the femur of a 6-year-old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase-PCR, Western blotting and flow cytometry. OS1 have aberrant G-banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS1 had ossification profiles similar to human fetal osteoblasts rather than SAOS-2 which ossifies ab initio (P < 0.05). Absence of p53 correlates with increased Runx2 expression, while the slow proliferation of OS1 cells is perhaps attenuated by pRB retention. OS1 express mesenchymal stem cell markers (CD44, CD105) and differ in relative expression of CD29, CD63, and CD71 to SAOS-2. (P < 0.05). Cell cycle synchronization with nocodazole did not affect Runx2 and CDK1 levels but decreased cyclin-E and increased cyclin-A (P < 0.05). Xenotransplantion of OS1 in SCID mice yields spontaneous tumors that were larger and grew faster than SAOS-2 transplants. Hence, OS1 is a new osteosarcoma cell culture model derived from a pre-chemotherapeutic ethnic Chinese patient, for mechanistic studies and development of therapeutic strategies to counteract metastasis and deregulation of mesenchymal development.

The p53‐mediated sensitivity of cancer cells to chemotherapeutic agents is conditioned by the status of the retinoblastoma protein

Despite the well-established function of p53 in determining cell cycle arrest and/or apoptosis in response to cytostatic/cytotoxic stresses, the role of the p53 status in the response to chemotherapeutic agents in human cancers has been not clearly defined. We wondered whether this was due to the fact that the p53-mediated response to chemotherapy drugs might be conditioned by the status of the retinoblastoma protein (pRb), a downstream factor of the pathway activated by p53 stabilization, which is frequently disrupted in cancer. The dependence of p53-mediated chemosensitivity on pRb status was first investigated in a prospective study on the prognostic relevance of p53 in breast cancer patients treated with adjuvant chemotherapy (5-fluorouracil, methotrexate and cyclophosphamide). Univariate analysis of disease-free survival (DFS) indicated that the p53 status, immunohistochemically evaluated, had no predictive value if considered independently of the pRb status. However, in patients with cancer with pRb neither lost nor hyperphosphorylated, p53 was significantly associated with the prognosis and, in a multivariate analysis of DFS including the established clinical and histopathological prognostic parameters, was found to be the only factor predicting the progression of the disease. We then studied the role of pRb status in the p53-mediated response to 5-fluorouracil and methotrexate or doxorubicin treatment in three human cancer cell lines. We found that in these cells the chemosensitivity was strictly dependent on the p53 status. However, either RB1 silencing or pRb hyperphosphorylation, caused by p16(INK4a) silencing, strongly reduced the p53-mediated response to chemotherapeutic agents. These results demonstrated that: (a) the p53-mediated response to chemotherapeutic agents induces a cytostatic/cytotoxic effect only in cancers with unaltered pRb pathway; and (b) the p53 status can actually predict the clinical outcome in this group of cancer patients.

Cellular growth inhibition by FK778 is linked to G1 arrest or S phase accumulation, dependent on the functional status of the retinoblastoma protein

The malononitrilamide FK778 is a novel immunosuppressive agent with antiproliferative activities. To gain insight into the molecular mechanism of FK778-mediated growth inhibition, we analyzed cells which differ in their p53 status and functionality of retinoblastoma protein (pRb). FK778 acted as a broad inhibitor of cell proliferation independent of the p53 or pRb status. However, the mechanism of FK778-mediated growth inhibition differed, leading either to cell cycle arrest in G1, or cell accumulation in S phase. This differential response was linked to the phosphorylation status of pRb. In addition, since FK778 was reported to exhibit antiviral activities, we analyzed the effect of FK778 on the growth stimulatory human papillomavirus (HPV)-16 and -18 E7 genes. Although growth of HPV-positive cells was strongly inhibited by FK778, we did not observe significant effects on viral E7 expression, indicating that the antiproliferative effect is not linked to an antiviral activity of FK778.

Cells enter a unique intermediate 4N stage, not 4N-G1, after aborted mitosis

It is widely accepted that mammalian cells enter the next G1-phase (G1) with 4N DNA after slippage from prolonged drug-induced mitotic block caused by activation of the transient spindle checkpoint. Understanding cell fate after mitotic slippage (MS) has significant clinical importance. The conclusion the MS cells enter 4N-G1 is based on morphology and mitotic cyclin destruction. Definitive biochemical evidence for G1 is scarce or unconvincing, in part because of methods of protein extraction required for immunoblot analysis that cannot take into account the cell cycle heterogeneity of cell cultures. We used single-cell-intracellular-flow-cytometric analysis to further define important factors determining cell fate after MS. Results from human and mouse embryonic stem cells (ESC) that reenter polyploid cell cycles are compared to human somatic cells that die after MS. We conclude that phosphorylation status of pRb, p53, CDK1, and especially cyclin B1 levels are important for cell fate decision in MS cells, which occur in a unique, intervening, non-G1, tetraploid subphase.

Molecular Mechanisms of Spindle Checkpoint-Apoptosis Linkage and Karyotypic Stability in Stem Cells.

Ex-vivo expansion of human HSC prior to bone marrow transplantation is still an unrealized goal that could greatly extend the usefulness of this mainstay strategy for treating numerous human hematologic diseases. The safety of this process for potential use in humans depends in large part on the maintenance of karyotypic stability of HSC during expansion, a lack of which could contribute to serious, even fatal, complications such as cancer, and could also contribute to engraftment failure. The spindle checkpoint and its linkage to apoptosis initiation is one of the most important cellular processes that helps maintain chromosomal stability in rapidly proliferating cell populations by removing aneuploid and karyotypically abnormal cells via activation of cell death programs. Detailed understanding of the molecular regulation of this vital cell cycle checkpoint is important to maximize safety of in-vitro HSC expansion techniques. It is widely accepted that mammalian cells enter the next G1-phase with 4N DNA after slippage from prolonged drug-induced mitotic block caused by activation of the transient spindle checkpoint that it is from this state that polyploid/aneuploid cells initiate apoptosis. However, definitive biochemical evidence for G1 is scarce or unconvincing; in part because of methods of protein extraction required for immunoblot analysis that cannot take into account the cell cycle heterogeneity of cell cultures. We used single-cell-intracellular-flow-cytometric analysis to define important factors determining cell fate after mitotic slippage. Results from human and mouse embryonic stem cells that reenter polyploid cell cycles are compared to human somatic hematopoietic cells that die after MS. We now report for the first time that phosphorylation status of pRb, p53, CDK1, and cyclin B1 levels are important for cell fate/apoptosis decision in mitotic-slippage cells, which occurs in a unique, intervening, non-G1, tetraploid subphase. Hyperphosphorylated Rb was extremely abundant in mitotic-slippage cells, a cell signaling event usually associated with early G1-S phase transition. P53 was phosphorylated at sites known to be associated with apoptosis regulation. Cyclin A and B1 were undetectable in mitotic slippage cells; yet, CDK1 was phosphorylated at sites typically associated with its activation. Evidence is also presented raising the possibility of cyclin B1-independent CDK1 activity in mitotic-slippage cells. These findings challenge the current models of spindle checkpoint-apoptosis linkages. Our new model could have important implications for methods to maintain karyotypic stability during ex-vivo HSC expansion.

Differential p38 mitogen-activated protein kinase-controlled hypophosphorylation of the retinoblastoma protein induced by nitric oxide in neuroblastoma cells

In this report we show that exogenous NO added to human neuroblastoma NB69 cells inhibits cell proliferation and downregulates the epidermal growth factor receptor (EGFR) and its downstream signaling pathways. These comprise the 3-phosphoinositide-dependent kinase 1/Akt/glycogen synthase kinase-3β pathway, the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinases 1 and 2 pathway, and the phospholipase Cγ pathway. In contrast, NO enhances the EGFR-controlled p38MAPK pathway. We also show that NO enhances the activation of the cAMP-responsive element binding protein, a transcription factor controlled by p38MAPK, as demonstrated using 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1 H-imidazole (SB202190), a p38MAPK inhibitor. These processes are accompanied by the NO-mediated hypophosphorylation of the retinoblastoma protein (pRb), preferentially at Ser795 compared to Ser780 and Ser807/811, and the downregulation of p27 KIP1, p21 CIP1/WAF1, and p16 INK4a, although NO downregulated p16 INK4a only when the p38MAPK activity was suppressed. The p38MAPK pathway controls the phosphorylation status of pRb as SB202190 enhances the hypophosphorylation of pRb. We reverted the inhibitory action of NO on EGFR and pRb phosphorylation in living cells using cell-permeable reducing agents, which suggested that reversible S-nitrosation controls these proteins. Our results support the notion that NO negatively modulates the p38MAPK-controlled phosphorylation of pRb, inducing the subsequent arrest of the cell cycle at the G1/S transition.

Promoter hypermethylation of TMS1, BRCA1, ERα and PRB in serum and tumor DNA of invasive ductal breast carcinoma patients

Breast cancer is fast emerging as the leading cancer amongst females, especially in younger age group in India; a large proportion of these tumors are often aggressive and ER and/or PR negative. Promoter methylation of genes involved in DNA repair and hormonal regulation may, in part, account for this behavior. To test this hypothesis methylation status of tumor suppressor genes TMS1, BRCA1, ERα and PRB was determined in invasive ductal carcinoma of breast and paired serum DNA. Of the 50 breast cancer patients investigated, 36/50 (72%) tumors and 32/50 (64%) paired sera showed methylation of at least one of these genes, while 17/50 (34%) tumors and 12/50 (24%) sera showed methylation of three genes. Methylation frequencies ranged from 24% for TMS1 to 63% for ERα. Significant association was observed between ERα and PRB methylation ( p ≤ 0.001) and there was concordance between DNA methylation in tumor and serum for each gene. Immunohistochemical analysis showed no detectable expression of ERα, PRB and BRCA1 in 21/36 (58%), 20/36 (56%) and 23/36 (64%) tumors respectively; significant correlation was observed between promoter methylation and loss of protein expression confirming our hypothesis that promoter methylation is an important mechanism for transcriptional silencing of these genes in breast cancer in this population. This study also underscores the potential utility of DNA methylation based screening of serum (a surrogate for tumor DNA methylation) as a tool for detection of breast cancer.

Predictive Value of Cell Cycle Biomarkers in Nonmuscle Invasive Bladder Transitional Cell Carcinoma

We determined whether the combined expression of p53, p21, p27 and pRB is associated with outcomes of patients with nonmuscle invasive bladder transitional cell carcinoma. Immunohistochemical staining for p53, p21, p27 and pRB was performed on archival bladder specimens from 9 normal controls and 74 patients who underwent transurethral bladder tumor resection for Ta, Tis and/or T1 transitional cell carcinoma. Normal urothelium had wild type status of p53, pRB, p21 and p27. p53 expression was altered in 34% of patients with transitional cell carcinoma, pRB in 39%, p21 in 35% and p27 in 47%. When analyzed separately, p53, pRB and p21 were each independently associated with tumor progression. Combination of biomarkers stratified patients into statistically significantly different risk groups for disease recurrence and progression. When tumor stage and grade were modeled with all 4 biomarkers, p53 and p27 were the sole independent predictors of disease recurrence and progression. After controlling for the effects of tumor grade and stage, the incremental number of altered biomarkers was associated with an increased risk of bladder cancer recurrence (p for trend = 0.011) and progression (p for trend = 0.005). The risk ratio for disease recurrence and progression increased incrementally with the number of biomarkers altered. Combinations of p53, pRB, p21 and p27 had cooperative/synergistic effects stratifying patients into different risk groups. Higher total numbers of altered biomarkers were independently associated with an increased risk of disease progression and death. Prospective trials are necessary to usher bladder cancer management into the age of molecular biomarkers.

RB acute loss induces centrosome amplification and aneuploidy in murine primary fibroblasts

BackgroundIncorrect segregation of whole chromosomes or parts of chromosome leads to aneuploidy commonly observed in cancer. The correct centrosome duplication, assuring assembly of a bipolar mitotic spindle, is essential for chromosome segregation fidelity and preventing aneuploidy. Alteration of p53 and pRb functions by expression of HPV16-E6 and E7 oncoproteins has been associated with centrosome amplification. However, these last findings could be the result of targeting cellular proteins in addition to pRb by HPV16-E7 oncoprotein. To get a more detailed picture on the role of pRb in chromosomal instability and centrosome amplification, we analyzed the effects of the acute loss of retinoblastoma gene function in primary conditional Rb deficient mouse embryonic fibroblasts (MEFs). Moreover, since pRb is a transcriptional repressor, microarray analysis was done on pRb-competent and pRb-deficient MEFs to evaluate changes in expression of genes for centrosome homeostasis and for correct mitosis.ResultsAcute loss of pRb induces centrosome amplification and aneuploidy in the vast majority of cells analyzed. A time course analysis shows a decrease of cells with amplified centrosomes after 40 days from the adenoviral infection. At this time only 12% of cells still show amplified centrosomes. Interestingly, cells with pRb constitutive loss show a similar percentage of cells with amplified centrosomes. DNA-Chip analyses in MEFs wt (mock infected) and pRb depleted (Ad-Cre infected) cells reveal differential expression of genes controlling both centrosome duplication and mitotic progression.ConclusionOur findings suggest a direct link between pRb status, centrosome amplification and chromosomal instability, and define specific mitotic genes as targets whose gene expression has to be altered to achieve or maintain aneuploidy.