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We developed a test to predict which patients will achieve pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) and which will have residual disease (RD). Gene expression data from pretreatment biopsies of patients with all breast cancer subtypes were combined into a 519-patient cohort containing 177 TNBC patients. Two RNA classifiers of 16 genes each were sequentially applied to the total cohort, classifying patients into 3 distinct classes. The test performance was further validated in an independent 304-patient cohort. The test accurately identified 70.5% (79/112) of pCR and 83.5% (340/407) of RD patients in the total population, and 75.0% (45/60) of pCR and 75.2% (88/117) of RD patients in the TNBC subset. For the independent cohort, the test identified 91.5% RD patients in the total population and 86.2% RD patients in the TNBC subset. However, the identification of pCR in both total and TNBC population are as low as 21.1% and 30%, respectively. The TNBC RD patients were subdivided by our classifiers, with one class showing significantly higher levels of Ki67 expression and having significantly poorer survival rates than the other classes. This stratification of patients may allow predicted residual disease classes to be assigned an alternative therapy.

Prediction of pathological complete response (pCR) for neoadjuvant treatment is an area of unmet clinical need, especially for triple negative breast cancer (TNBC) as pCR is correlated with better outcomes. Predicting which patients will have residual disease (RD) provides an opportunity to improve treatment planning. We developed a test to predict which patients are likely to achieve pCR or RD to the standard of care (taxane-based) neoadjuvant chemotherapy using gene expression profiling of 325 previously identified novel biomarkers. Three microarray datasets were used (GSE22226, GSE25055, and GSE25065) including a total of 594 stage II-III breast cancer patients of which 125 (21%) achieved pCR, and 469 (79%) RD. ER+ tumors were present in 57% of the patients and 52% were PGR+. Almost 90% of the patients were Her2-. Of 231 TNBC, 78 (33.8%) achieved pCR, while 153 (66.2%) RD. Of 303 ER+Her- patients 26 (8.6%) achieved pCR while 277 (91.4%) RD. The cohort was divided into balanced populations with 476 patients used for training (80%) and test (20%) rounds of model development, while 118 patients were reserved as a validation set. Combining a “winnowing” process to remove genes with least predictive power, and hundreds of thousands of step-wise runs, followed by ranking genes based on conditional probabilities, we developed a 17-gene cassette (BA100) which was locked-down in the validation set with ROC (AUC) = 0.818. With a cut-off of 83% sensitivity and 68% specificity (PPV 0.4; NPV 0.94), BA100 achieved a 16% true positive rate (true pCR) and 55% true negative rate (true RD) identifying 76% of the patients who achieved pCR, and 69% of the patients with RD. In TNBC, BA100 classified 29% as true positives (TP), 36% as false positive (FP), 30% true negative (TN), and 4.8% false negative (FN). Kaplan Meier (KM) curves showed a significant difference in 5-year disease-free survival (5Y DFS) between TP and TN (p=0.00453) or FP (p=2.09E-06). However, FP had even worse outcomes than TN patients. To improve the TP rate, additional genes expressed in TNBC plus the original 325 genes were subjected to a second round of gene selection to discriminate between TP and FP, resulting in a 16-gene cassette (BA100.1). With a cut-off of 95% sensitivity and 73% specificity (PPV 0.7; NPV 0.95), applying BA100.1 reduced the FP rates from 24% to 9%, while correctly identifying 88% of RD in the validation set. KM curves showed no significant difference in 5Y DFS between 124 TNBC (53.7%) classified as TN versus 29 TNBC (12.6%) classified as FP, while a significant difference in survival rate was found between TNBC classified as TN vs TP (Cox Proportional Harzard p=8.42e-05). Taken together, we developed a predictive test consisting of two gene cassettes that accurately identified 71% (88/104) of pCR, and 88% (417/469) of RD patients. Gene cassettes include several transcriptional repressors, PI3K signal transduction, components of telomerase, DNA repair genes, fatty acid metabolism and estrogen-independent proliferation. The test stratified TNBC with differential response to chemotherapy and survival rates so that novel approaches can be used without delay. Further validation will confirm the test utility. Citation Format: Fournier MV, Chen J, Obenauer J, Goodwin EC, Tannenbaum SH, Brufsky AM. A predictive test for neoadjuvant chemotherapy in breast cancer identifies a subset of triple negative patients with resistant disease and the poorest prognosis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-10-08.

Background. With the development of new drug combinations and targeted treatments for multiple types of cancer, the ability to stratify categories of patient populations and to develop companion diagnostics has become increasingly important. A panel of 325 RNA biomarkers was selected based on cancer-related biological processes of healthy cells and gene expression changes over time during nonmalignant epithelial cell organization. This “cancer in reverse” approach resulted in a panel of biomarkers relevant for at least 7 cancer types, providing gene expression profiles representing key cellular signaling pathways beyond mutations in “driver genes.” Objective. To further investigate this biomarker panel, the objective of the current study is to (1) validate the assay reproducibility for the 325 RNA biomarkers and (2) compare gene expression profiles side by side using two technology platforms. Methods and Results. We have mapped the 325 RNA transcripts and in a custom NanoString nCounter expression panel to be compared to all potential probe sets in the Affymetrix Human Genome U133 Plus 2.0. The experiments were conducted with 10 unique biological formalin-fixed paraffin-embedded (FFPE) breast tumor samples. Each site extracted RNA from four sections of 10-micron thick FFPE tissue over three different days by two different operators using an optimized standard operating procedure and quality control criteria. Samples were analyzed using mas5 in BioConductor and NanoStringNorm in R. Pearson correlation showed reproducibility between sites for all 60 samples with r=0.995 for Affymetrix and r=0.999 for NanoString. Correlation in multiple days and multiple users was for Affymetrix r=0.962−0.999 and for NanoString r=0.982−0.991. Conclusion. The 325 RNA biomarkers showed reproducibility in two technology platforms with moderate to high concordance. Future directions include performing clinical validation studies and generating rationale for patient selection in clinical trials using the technically validated assay.

High-risk human papillomaviruses (HPVs) cause a variety of malignancies of the mucosal epithelium. However, the local immune evasion strategies used by HPV-transformed cells remain unclear. Here, we examined the effect of HPV-positive cancer cells on human peripheral blood monocytes, which are precursors of Langerhans cells, key antigen-presenting cells in the squamous epithelium. HPV-positive cervical cancer cells and HPV-E6 expressing cells inhibited monocyte differentiation to Langerhans cells in a contact-dependent manner. Unlike Langerhans cells, monocytes that differentiated in the presence of HPV16 E6-expressing cells exhibited high levels of endocytic activity. Our results suggest that cells infected by high-risk HPV evade immune surveillance by blocking the differentiation of monocytes into competent antigen presenting cells.

Screening and prevention is critical to reducing breast cancer deaths, and mammography is not an ideal tool. Mammography has not reduced invasive breast cancer incidence, it has high numbers of false positives and it detects many non-invasive cancers that are not life threatening. Metastatic breast cancer is the second leading cause of cancer death for women. The development of a new blood test for breast cancer screening could offer significant improvements with the potential to be more accurate, less costly, less intimidating, not subject to age differences, and able to discriminate between cancers subtypes. Detection of breast cancer cells (BCC) in blood samples offers a valuable opportunity for screening. However, BCC in blood are rare and must be identified against a background of millions of other blood cells. Although many technologies have been developed for the isolation and detection of circulating tumor cells using EpCAM and cytokeratins, these tests do not provide levels of sensitivity and specificity required for a cancer screening tool. To address this critical unmet need, we developed a panel of cell surface markers whose expression patterns specifically distinguish breast cancer cells in blood samples. These genes were selected based on a discovery platform tailored to detect early stage breast cancers and include 20 novel putative cell surface molecules that are candidates for breast cancer cell detection and isolation. We prioritized these molecules by specificity of expression in tumor cells including: 1) Ratio >25:1 average breast tumor biopsy expression versus average normal blood expression, 2) High expression relative (>3 fold) in >20% of tumor biopsy samples, 3) High expression (>3 fold) in >40% of breast cancer cell lines, and 4) Expression significantly associated with 10-year outcomes (Kaplan Meier analysis, p Citation Format: Lucas Delmonico, Edward C. Goodwin, Marcia Vasconcellos Fournier. Isolation and detection of breast cancer cells in blood samples using antibody cocktails selected by large-scale genome-wide screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5576.

From over two hundred driver mutations identified to date, only about a dozen are FDA-approved biomarkers, and there is an unmet need to discover novel suitable biomarkers. We selected novel biomarkers based on non-malignant breast epithelial cell phenotypes and identified 325 genes (BA325) showing 32 significant oncology drug associations. A total of 251 genes out of 325 are unique and not found in any of 9 other oncology panels investigated, suggesting that BA325 may yield novel insights regarding tumor biology, clinical outcomes, and novel therapeutic targets, not covered by current tools. While prior work has validated the utility of BA325 in breast cancer, the current study investigates BA325 expression in other tumor types beyond breast. We tested BA325 expression in 8 tumor types (breast, colon, lung, ovarian, prostate, pancreatic, gastric cancers, and leukemia), using two independent public data sets for each, totaling 3,563 samples in 16 datasets. All used Affymetrix HG-U133A or Plus 2.0 microarrays. Samples were normalized within each study using RMA and batch-corrected across studies. BA325 expression levels were investigated, showing at least 324 of the 325 genes expressed above background levels (log2 > 4) in all cases tested. The expression profile of BA325 differed in magnitude and pattern across tumor types from 1,000 randomly generated sets of 325 genes. Surprisingly, 102 of the BA325 genes were among 3,804 published housekeeping (HK) genes, with 7 of them present in a predictive signature developed for breast cancer chemotherapy response. The results suggest HK genes may play a role in the underlying biology of how cancer cells respond to treatments. A set of 119 genes showed tissue-specific expression (10 breast, 2 colon, 7 lung, 3 ovarian, and 42 prostate), while 87 genes showed the least variation across tumors (4-fold change or less), including the genes MPRIP, MUS81, and AKT1. Twenty seven (27) of the tissue-specific genes and 48 of the least-varying genes are also reported to be HK genes. Differential expression was tested by unsupervised clustering in 14 of the 16 studies. BA325 genes successfully distinguished tumor and normal samples in gastric and ovarian cancers, and subtypes of lung and breast cancers, with implications for treatment responses. We conclude that BA325 expression profiles in all datasets examined include both tissue-specific genes and genes with similar expression across tissues. Preliminary results indicate BA325 genes may have utility as biomarkers in a surprisingly wide variety of tumor types (including leukemia) in addition to breast cancer, with discriminatory power in at least gastric, ovarian, lung and breast cancers. Thus, BA325 can greatly increase the biomarker repertoire beyond oncogenes or other driver genes and may provide relevant insight in novel oncology therapeutic targets. Citation Format: Marcia V. Fournier, John C. Obenauer, Andreia Maer, Edward C. Goodwin. Genes involved in non-malignant breast phenotypes are widely expressed in multiple cancers and provide novel biomarkers of clinical outcomes and therapeutic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-217.

We developed a high-throughput, cell-based screen to identify chemicals that inhibit infection by the primate polyomaviruses. The screen is based on the detection of compounds that inhibit the ability of a replication-defective simian virus 40 (SV40)-based viral vector to cause growth arrest in HeLa cells by repressing the expression of the endogenous human papillomavirus E7 oncogene in these cells. We identified two compounds, ellagic acid and spiperone, that suppressed the ability of the SV40 recombinant virus to inhibit cellular DNA synthesis. These compounds caused a marked reduction of the ability of wild-type SV40 to productively infect permissive monkey cells, even when the compounds were added several hours after infection. The fraction of cells expressing SV40 large T antigen and the levels of T antigen mRNA were reduced in infected human and monkey cells treated with ellagic acid and spiperone, suggesting that these compounds block a step in the virus life cycle prior to SV40 early gene expression. Ellagic acid and spiperone also inhibited large T antigen expression by BK virus and JC virus, two important, pathogenic human polyomaviruses.

Apoptosis is a potent host defense against microbes. Most viruses have adapted strategies to counteract this response. Herpes simplex virus (HSV) produces a balance between pro- and antiapoptotic processes during infection. When antiapoptotic signals become limiting, infected cells die through HSV-dependent apoptosis (HDAP). Oncogenic pathways were previously implicated in HDAP susceptibility. Here, we exploited our ability to selectively express all, one, or no oncogenes in the well-defined HeLa cell system to dissect the requirements for HDAP. Human papillomavirus E6 and E7 oncogene expression was inhibited by the E2 viral repressor. Sole expression of E6 mediated HDAP sensitization. Next, two known cellular targets of E6 were independently modulated. This demonstrated that E6 sensitizes HeLa cells to HDAP through hTERT and p53. Given the universality of the apoptotic antiviral response, p53 and telomerase regulation will likely be important for counteracting host defenses in many other viral infections.

This work demonstrates a central role for the retinoblastoma (Rb) family in driving the transcriptional program of induced and replicative senescence. HeLa cervical carcinoma cells rapidly undergo senescence when the human papillomavirus (HPV) type 18 E7 gene in these cells is repressed by the bovine papillomavirus (BPV) E2 protein. This senescence response requires the endogenous Rb pathway but not the p53 pathway. Microarray analysis 6 days after BPV E2 introduction into HeLa cells identified 224 cellular genes induced by E7 repression and 354 repressed genes. Many repressed genes were involved in cell cycle progression, and numerous induced genes encoded lysosomal proteins. These gene expression changes were blocked by constitutive expression of the wild-type HPV16 E7 or adenovirus E1A gene, but not by E7 or E1A mutants defective for Rb binding. Short hairpin RNAs targeting the Rb family also inhibited these gene expression changes and blocked senescence. Therefore, surprisingly, the transcriptional response to BPV E2 expression was entirely dependent on E7 repression and activation of the Rb family, and the BPV E2 protein did not directly affect the expression of cellular genes. Activation of the Rb family repressed E2F-responsive genes and stimulated transcriptional activators, thereby mobilizing multiple signals, such as repression of B-MYB and DEK, that were independently sufficient to induce senescence. There was extensive overlap between the transcriptional profiles of senescent, late-passage primary human fibroblasts and senescent cervical carcinoma cells, suggesting that this Rb family-mediated transcriptional cascade also plays a central role in replicative senescence.

Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm., Author Summary The human papillomaviruses are important carcinogens, but little is known about how these non-enveloped viruses traffic to the nucleus, the site of genome replication. We use imaging, biochemical, and genetic techniques to show that a multi-subunit intracellular trafficking machine known as retromer binds directly to the L2 minor capsid protein in the virus particle to initiate its transport from the endosome to other membrane-bound organelles farther inside the cell. Most notably, knock-down of retromer expression or mutation of newly identified retromer binding sites in L2 cause the accumulation of incoming HPV16 capsids in the endosome and prevent trafficking to the Golgi. These defects can be corrected by insertion of a retromer binding site from a cellular cargo. Because all previously known retromer cargoes are cellular transmembrane proteins, the virus represents a new class of retromer cargo. In addition to elucidating the mechanism of viral endosome escape, these results suggest that retromer may play a more versatile role in cell biology than previously recognized.

Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins, and repression of HPV gene expression causes the cells to cease proliferation and undergo senescence. However, it is not known whether both HPV proteins are required to maintain the proliferative state of cervical cancer cells, or whether mutations that accumulate during carcinogenesis eliminate the need for one or the other of them. To address these questions, we used the bovine papillomavirus E2 protein to repress the expression of either the E6 protein or the E7 protein encoded by integrated HPV18 DNA in HeLa cervical carcinoma cells. Repression of the E7 protein activated the Rb pathway but not the p53 pathway and triggered senescence, whereas repression of the E6 protein activated the p53 pathway but not the Rb pathway and triggered both senescence and apoptosis. Telomerase activity, cyclin-dependent kinase activity, and expression of c-myc were markedly inhibited by repression of either E6 or E7. These results demonstrate that continuous expression of both the E6 and the E7 protein is required for optimal proliferation of cervical carcinoma cells and that the two viral proteins exert distinct effects on cell survival and proliferation. Therefore, strategies that inhibit the expression or activity of either viral protein are likely to inhibit the growth of HPV-associated cancers.

Background: We developed an algorithm based on the gene expression of tumor biopsies to identify the best combination of biomarkers to reliably predict a patient’s response to relevant cancer treatments. This algorithm is derived from 325 genes whose expression showed significant changes during differentiation of non-malignant human mammary epithelial cells cultured in laminin-rich extracellular matrix. Of these 325 genes, 251 are novel and not present in 9 other cancer based gene expression panels such as FoundationOne or PAM50. These differentiated cells formed multicellular structures with defined lumens and tight junctions and with specific localizations of cadherin and integrins. In contrast, cells from breast cancers displayed a general loss of structure. Previous work showed that different sets of these 325 biomarkers accurately predicted overall breast cancer patients' survival or response to neoadjuvant therapy in multiple independent studies. Objective: Predict cell-signaling pathways, drug associations, and disease associations for the 325 biomarkers (BA325) in contrast to other cancer gene panels. Methods and Results: The Qiagen Ingenuity program was used to identify pathways and disease states containing significant overlap with BA325. Multiple cell signaling pathways including cell proliferation, migration, invasion, and metabolism were found in BA325, while most other cancer biomarker panels were highly concentrated in cell proliferation. Examples of significant pathway associations relevant for oncology drug discovery and targeted treatments include Cell Cycle Control of Chromosomal Replication (p=8.1E-14), Polo-like-kinase and HSP90 complex (p=6.3E-07), G2/M DNA Damage Checkpoint (p=6.4E-07), Integrin Signaling (p=3E-05), Integrin Linked Kinase Signaling (p=4.51E-05), BRCA1 DNA Damage Response (p=1.75E-04), Estrogen Mediated S-phase entry (p=4.24E-04), Regulation of Actin Based Motility by RHO (p=4.55E-04), Adherins Junction Remodeling (p=5.6E-04), Actin Cytoskeleton Signaling (p=7.25E-04), Mismatch Repair (p=1.6E-03), RAN Signaling (p=1.64E-03), Protein Ubiquitination (p=1.78E-03) and Cholesterol Biosynthesis (p=4.8E-03). This wide coverage of cellular signaling pathways provides an advantage in developing predictive tests for treatments targeting highly complex cell signaling networks. Surprisingly, even though the BA325 panel was obtained during non-malignant differentiation, the most significant disease state associated with these genes was mammary tumor, demonstrating the validity of this approach to probe breast cancer biology. Conclusion: This analysis demonstrates that the BA325 panel is useful both in understanding non-malignant mammary epithelial differentiation and breast cancer tumors. Citation Format: Edward C. Goodwin, Said Attiya, Marcia Fournier. A novel panel of 325 biomarkers is part of a large interconnected network representing multiple cell signaling pathways and allowing development of predictive tests for oncology drugs [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 405. doi:10.1158/1538-7445.AM2017-405

DNAJB12 and DNAJB14 are transmembrane proteins in the endoplasmic reticulum (ER) that serve as co-chaperones for Hsc70/Hsp70 heat shock proteins. We demonstrate that over-expression of DNAJB12 or DNAJB14 causes the formation of elaborate membranous structures within cell nuclei, which we designate DJANGOS for DNAJ-associated nuclear globular structures. DJANGOS contain DNAJB12, DNAJB14, Hsc70 and markers of the ER lumen and ER and nuclear membranes. Strikingly, they are evenly distributed underneath the nuclear envelope and are of uniform size in any one nucleus. DJANGOS are composed primarily of single-walled membrane tubes and sheets that connect to the nuclear envelope via a unique configuration of membranes, in which the nuclear pore complex appears anchored exclusively to the outer nuclear membrane, allowing both the inner and outer nuclear membranes to flow past the circumference of the nuclear pore complex into the nucleus. DJANGOS break down rapidly during cell division and reform synchronously in the daughter cell nuclei, demonstrating that they are dynamic structures that undergo coordinate formation and dissolution. Genetic studies showed that the chaperone activity of DNAJ/Hsc70 is required for the formation of DJANGOS. Further analysis of these structures will provide insight into nuclear pore formation and function, activities of molecular chaperones, and mechanisms that maintain membrane identity.

The papillomavirus E2 proteins can function as sequence-specific transactivators or transrepressors of transcription and as cofactors in viral DNA replication. We previously demonstrated that acute expression of the bovine papillomavirus type 1 (BPV1) E2 protein in HeLa and HT-3 cervical carcinoma cell lines greatly reduced cellular proliferation by imposing a specific G 1 /S phase growth arrest. In this report, we analyzed the effects of a panel of point mutations in the BPV1 E2 protein to identify the functional requirements for acute growth inhibition. Disruption of E2-specific transactivation by mutations within either the transactivation domain or the DNA binding domain severely impaired E2-mediated growth inhibition in HeLa and HT-3 cells, even though these mutants retain various other E2 activities. This result indicates that functional transactivation activity is required for acute E2-mediated growth inhibition. HeLa cells, which contain a wild-type p53 gene, and HT-3 cells, which contain a transactivation-defective p53 gene, exhibited similar responses to the E2 mutants, suggesting that identical functions of the E2 protein were required for growth arrest regardless of p53 status. Replacement of the E2 transactivation domain with that of the herpes simplex virus VP16 generated a chimeric transactivator that efficiently stimulated expression of an E2-responsive reporter plasmid yet was completely defective for growth inhibition, suggesting that an E2-specific transactivation function is required for growth arrest. Surprisingly, the transactivation-defective E2 mutants were also markedly defective in their ability to repress transcription of the native human papillomavirus type 18 (HPV18) E6/E7 oncogenes in HeLa cells and of the HPV18 promoter present in a transfected reporter plasmid. These mutants were also defective in their ability to increase p53 levels. Therefore, efficient repression of the HPV18 promoter in HeLa cells is not merely a consequence of the binding of an E2 protein to appropriately situated binding sites in the promoter.

Simian virus 40 (SV40) is a nonenveloped DNA virus that traffics through the endoplasmic reticulum (ER) en route to the nucleus, but the mechanisms of capsid disassembly and ER exit are poorly understood. We conducted an unbiased RNA interference screen to identify cellular genes required for SV40 infection. SV40 infection was specifically inhibited by up to 50-fold by knockdown of four different DNAJ molecular cochaperones or by inhibition of BiP, the Hsp70 partner of DNAJB11. These proteins were not required for the initiation of capsid disassembly, but knockdown markedly inhibited SV40 exit from the ER. In addition, BiP formed a complex with SV40 capsids in the ER in a DNAJB11-dependent fashion. These experiments identify five new cellular proteins required for SV40 infection and suggest that the binding of BiP to the capsid is required for ER exit. Further studies of these proteins will provide insight into the molecular mechanisms of polyomavirus infection and ER function., IMPORTANCE The polyomaviruses, including simian virus 40 (SV40), are important human pathogens and model systems for exploring the general features of virus replication and cell biology. We used a genetic system to interrogate the role of cellular genes in SV40 infection. Based on the results of this unbiased genetic screen and analysis of proteins related to the strongest hit from the screen, we identified five new cellular proteins required for the entry of SV40 into cells. These proteins physically associate with SV40 in the endoplasmic reticulum (ER) during virus entry and are required for exit of the partially disassembled virus from this organelle. These results demonstrate that the polyomaviruses have coopted an ER-localized protein quality control process to initiate disassembly and transit through the cell on their way to the nuclear site of virus replication.

Replicative senescence limits the proliferation of somatic cells passaged in culture and may reflect cellular aging in vivo. The most widely used biomarker for senescent and aging cells is senescence-associated beta-galactosidase (SA-beta-gal), which is defined as beta-galactosidase activity detectable at pH 6.0 in senescent cells, but the origin of SA-beta-gal and its cellular roles in senescence are not known. We demonstrate here that SA-beta-gal activity is expressed from GLB1, the gene encoding lysosomal beta-D-galactosidase, the activity of which is typically measured at acidic pH 4.5. Fibroblasts from patients with autosomal recessive G(M1)-gangliosidosis, which have defective lysosomal beta-galactosidase, did not express SA-beta-gal at late passages even though they underwent replicative senescence. In addition, late passage normal fibroblasts expressing small-hairpin interfering RNA that depleted GLB1 mRNA underwent senescence but failed to express SA-beta-gal. GLB1 mRNA depletion also prevented expression of SA-beta-gal activity in HeLa cervical carcinoma cells induced to enter a senescent state by repression of their endogenous human papillomavirus E7 oncogene. SA-beta-gal induction during senescence was due at least in part to increased expression of the lysosomal beta-galactosidase protein. These results also indicate that SA-beta-gal is not required for senescence.

Cervical cancer arises from lesions caused by infection with high-risk types of human papillomavirus (HPV). Therefore, vaccination against HPV could prevent carcinogenesis by preventing HPV infection or inducing lesion regression. HPV E2 protein is an attractive candidate for vaccine development because it is required for papilloma formation, is involved in all stages of the virus life cycle, and is expressed in all premalignant lesions as well as some cancers. This study reports vaccination against E2 protein using a rabbit model of papillomavirus infection. A recombinant adenovirus (Ad) vector expressing the E2 protein of cottontail rabbit papillomavirus (CRPV) was tested for therapeutic efficacy in CRPV-infected rabbits. Primary immunization with the Ad-E2 vaccine, compared to immunization with a control Ad vector, reduced the number of papilloma-forming sites from 17 of 45 to 4 of 45. After booster immunization, vaccinated rabbits formed no new papillomas versus an additional 23 papillomas in rabbits that received the control vector. Papillomas in the Ad-E2 vaccinees were significantly smaller than those in the control rabbits, and all four papillomas in the Ad-E2 vaccinated rabbits regressed. No CRPV DNA was detected either in the regression sites or in sites that did not form papillomas, indicating that the vaccination led to clearance of CRPV from all infected sites.

Most cervical carcinomas express high-risk human papillomaviruses (HPVs) E6 and E7 proteins, which neutralize cellular tumor suppressor function. To determine the consequences of removing the E6 and E7 proteins from cervical cancer cells, we infected HeLa cells, a cervical carcinoma cell line that contains HPV18 DNA, with a recombinant virus that expresses the bovine papillomavirus E2 protein. Expression of the E2 protein resulted in rapid repression of HPV E6 and E7 expression, followed ≈12 h later by profound inhibition of cellular DNA synthesis. Shortly after E6/E7 repression, there was dramatic posttranscriptional induction of p53. Two p53-responsive genes, mdm2 and p21, were induced with slightly slower kinetics than p53 and appeared to be functional, as assessed by inhibition of cyclin-dependent kinase activity and p53 destabilization. There was also dramatic posttranscriptional induction of p105 Rb and p107 after E6/E7 repression, followed shortly thereafter by induction of p130. By 24 h after infection, only hypophosphorylated p105 Rb was detectable and transcription of several Rb/E2F-regulated genes was dramatically repressed. Constitutive expression of the HPV16 E6/E7 genes alleviated E2-induced growth inhibition and impaired activation of the Rb pathway and repression of E2F-responsive genes. This dynamic response strongly suggests that the p53 and Rb tumor suppressor pathways are intact in HeLa cells and that repression of HPV E6 and E7 mobilizes these pathways in an orderly fashion to deliver growth inhibitory signals to the cells. Strikingly, the major alterations in the cell cycle machinery underlying cervical carcinogenesis can be reversed by repression of the endogenous HPV oncogenes.

Expression of the bovine papillomavirus E2 regulatory protein in human cervical carcinoma cell lines repressed expression of the resident human papillomavirus E6 and E7 oncogenes and within a few days caused essentially all of the cells to synchronously display numerous phenotypic markers characteristic of cells undergoing replicative senescence. This process was accompanied by marked but in some cases transient alterations in the expression of cell cycle regulatory proteins and by decreased telomerase activity. We propose that the human papillomavirus E6 and E7 proteins actively prevent senescence from occurring in cervical carcinoma cells, and that once viral oncogene expression is extinguished, the senescence program is rapidly executed. Activation of endogenous senescence pathways in cancer cells may represent an alternative approach to treat human cancers.

Prolactin and growth hormone are members of a gene family which has been extensively studied during the past several years (Miller and Eberhardt, 1983). Along with the gonadotropins, the members of this gene family are primarily expressed in the anterior pituitary. The expression of these genes and their corresponding regulatory factors has been of interest from the perspective of differential regulation during developmental stages, hormonal effects on gene expression, and the possibility of expression at ectopic sites other than the pituitary (Gluckman et al., 1981). Studies described in this manuscript are restricted to the growth hormone and prolactin genes and more specifically to aspects of gene structure which influence the level of their respective mRNAs.

Expression of the bovine papillomavirus E2 protein in cervical carcinoma cells represses expression of integrated human papillomavirus (HPV) E6/E7 oncogenes, followed by repression of the cdc25A gene and other cellular genes required for cell cycle progression, resulting in dramatic growth arrest. To explore the mechanism of repression of cell cycle genes in cervical carcinoma cells following E6/E7 repression, we analyzed regulation of the cdc25A promoter, which contains two consensus E2F binding sites and a consensus E2 binding site. The wild-type E2 protein inhibited expression of a luciferase gene linked to the cdc25A promoter in HT-3 cervical carcinoma cells. Mutation of the distal E2F binding site in the cdc25A promoter abolished E2-induced repression, whereas mutation of the proximal E2F site or the E2 site had no effect. None of these mutations affected the activity of the promoter in the absence of E2 expression. Expression of the E2 protein also led to posttranscriptional increase in the level of E2F4, p105(Rb), and p130 and induced the formation of nuclear E2F4-p130 and E2F4-p105(Rb) complexes. This resulted in marked rearrangement of the protein complexes that formed at the distal E2F site in the cdc25A promoter, including the replacement of free E2F complexes with E2F4-p105(Rb) complexes. These experiments indicated that repression of E2F-responsive promoters following HPV E6/E7 repression was mediated by activation of the Rb tumor suppressor pathway and the assembly of repressing E2F4-Rb DNA binding complexes. Importantly, these experiments revealed that HPV-induced alterations in E2F transcription complexes that occur during cervical carcinogenesis are reversed by repression of HPV E6/E7 expression.