Platelet-derived growth factor induces apoptosis in growth-arrested murine fibroblasts.

The platelet-derived growth factor (PDGF) is a potent mitogen for murine fibroblasts. PDGF-stimulated cells express a set of immediate-early-response genes but require additional (progression) factors in serum to progress through the cell cycle. Serum-deprived cells are reversibly arrested in G0 phase and fail to fully traverse the G1 phase of the cell cycle when stimulated by PDGF alone. We now report that serum-deprived normal rat kidney fibroblast (NRK) cells stimulated by either PDGF AA or PDGF BB homodimers undergo apoptotic cell death. Furthermore, we show that epidermal growth factor also induces apoptotic cell death in serum-deprived NRK cells, epidermal growth factor enhances the rate of apoptosis in PDGF-treated cells, and a progression factor (insulin) but not endogenously expressed Bc1-2 fully protects NRK cells from PDGF-stimulated apoptosis. The results indicate that PDGF induces apoptosis in growth-arrested NRK cells and that the inability of NRK cells to transit the G1/S checkpoint is the critical determinant in establishing the genetic program(s) to direct the PDGF signal to apoptosis. The results suggest that polypeptide growth factors in vivo may signal cell fate positively or negatively in settings that limit the potential of cells to completely transit the cell cycle.

Basic fibroblast growth factor can mediate the early inductive events in renal development.

The earliest characterized events during induction of tubulogenesis in renal anlage include the condensation or compaction of metanephrogenic mesenchyme with the concurrent upregulation of WT1, the gene encoding the Wilms tumor transcriptional activator/suppressor. We report that basic fibroblast growth factor (FGF2) can mimic the early effects of an inductor tissue by promoting the condensation of mesenchyme and inhibiting the tissue degeneration associated with the absence of an inductor tissue. By in situ hybridization, FGF2 was also found to mediate the transcriptional activation of WT1 and of the hepatocyte growth factor receptor gene, c-met. Although FGF2 can induce these early events of renal tubulogenesis, it cannot promote the epithelial conversion associated with tubule formation in metanephrogenic mesenchyme. For this, an undefined factor(s) from pituitary extract in combination with FGF2 can cause tubule formation in uninduced mesenchyme. These findings support the concept that induction in kidney is a multiphasic process that is mediated by more than a single comprehensive inductive factor and that soluble molecules can mimic these inductive activities in isolated uninduced metanephrogenic mesenchyme.

Polymerizable Peptide Monomers for the Targeted and Intracellular Delivery of Cancer Therapeutics

Thesis (Ph.D.)--University of Washington, 2016-06

Nuclear translocation of cell-surface receptors: Lessons from fibroblast growth factor

The nuclear localization of a number of growth factors, cytokine ligands and their receptors has been reported in various cell lines and tissues. These include members of the fibroblast growth factor (FGF), epidermal growth factor and growth hormone families. Accordingly, a number of nuclear functions have begun to emerge for these protein families. The demonstration of functional interactions of these proteins with the nuclear import machinery has further supported their functions as nuclear signal transducers. Here, we review the membrane- trafficking machinery and pathways demonstrated to regulate this cell surface to nucleus-trafficking event and highlight the many remaining unanswered questions. We focus on the FGF family, which is providing many of the clues as to the process of this unusual phenomenon.

Evaluation of oestrogen and progesterone receptor status in HER-2 positive breast carcinomas and correlation with outcome

Aim: HER-2/neu amplification occurs in 15-25% of breast carcinomas. This oncogene, also referred to as c-erbB-2, encodes a transmembrane tyrosine kinase receptor belonging to the epidermal growth factor receptor family. HER-2 over-expression is reported to be associated with a poor prognosis in breast carcinoma patients and in some studies is associated with a poorer response to anti-oestrogen therapy. These patients are less likely to benefit from CMF (cyclophosphamide, methotrexate, fluorouracil)-based chemotherapy compared with anthracycline-based chemotherapy. The aim of this study was to evaluate breast carcinomas to determine hormone receptor status and if there is a difference in breast cancer specific survival for HER-2 positive patients. Methods: A total of 591 breast carcinomas were evaluated using immunohistochemistry (IHC) for oestrogen receptor (ERp), progesterone receptor (PRp) and three different HER2 antibodies (CB11, A0485 and TAB250). Percentage of tumour cells and intensity of staining for ERp were evaluated using a semiquantitative method. Results: Of the 591 tumours, 91 (15.4%) showed 3+ membrane staining for HER-2 with one or more antibodies. Of these 91 tumours, 41 (45.1%) were ERp+/ PRp+, seven (7.7%) were ERp+/PR-, six (6.6%) were ERp-/PRp+ and 37 (40.7%) were ERp-/PR-. Of HER-2 positive tumours, 5.5% showed > 80% 3+ staining for ERp compared with 31.8% of 0-2+ HER-2 tumours; 24.2% of HER-2-positive tumours showed 60% or more cells with 2+ or 3+ staining for ERp. Treatment data were available for 209 patients and no difference was observed in breast cancer specific survival (BCSS) with HER-2 status and tamoxifen. Conclusion: Oestrogen receptor status cannot be used to select tumours for evaluation of HER-2 status, and oestrogen and progesterone receptor positivity does not preclude a positive HER-2 status. There is a higher proportion of ERp negative tumours associated with HER-2 positivity, however, more than 20% of HER-2 positive tumours show moderate or strong staining for ERp. HER-2 positive patients in this study did not show an adverse BCSS with tamoxifen treatment unlike some previous studies.

Sequence and chemistry requirements for a novel aptameric oligonucleotide inhibitor of EGF receptor tyrosine kinase activity

We have previously identified a phosphorothioate oligonucleotide (PS-ODN) that inhibited epidermal growth factor receptor tyrosine kinase (TK) activity both in cell fractions and in intact A431 cells. Since ODN-based TK inhibitors may have anti-cancer applications and may also help understand the non-antisense mediated effects of PS-ODNs, we have further studied the sequence and chemistry requirements of the parent PS-ODN (sequence: 5′-GGA GGG TCG CAT CGC-3′) as a sequence-dependent TK inhibitor. Sequence deletion and substitution studies revealed that the 5′-terminal GGA GGG hexamer sequence in the parent compound was essential for anti-TK activity in A431 cells. Site-specific substitution of any G with a T in this 5′-terminal motif within the parent compound caused a significant loss in anti-TK activity. The fully PS-modified hexameric motif alone exhibited equipotent activity as the parent 15-mer whereas phosphodiester (PO) or 2′-O-methyl-modified versions of this motif had significantly reduced anti-TK activity. Further, T substitutions within the two 5′-terminal G residues of the hexameric PS-ODN to produce a sequence, TTA GGG, representing the telomeric repeats in human chromosomes, also did not exhibit a significant anti-TK activity. Multiple repeats of the active hexameric motif in PS-ODNs resulted in more potent inhibitors of TK activity than the parent ODN. These results suggested that PS-ODNs, but not PO or 2′-O-methyl modified ODNs, containing the GGA GGG motif can exert potent anti-TK activity which may be desirable in some anti-tumor applications. Additionally, the presence of this previously unidentified motif in antisense PS-ODN constructs may contribute to their biological effects in vitro and in vivo and should be accounted for in the design of the PS-modified antisense ODNs. © 2002 Published by Elsevier Science Inc.

X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity.

Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.

Focused molecular analysis of small cell lung cancer: feasibility in routine clinical practice.

Background: There is an urgent need to identify molecular signatures in small cell lung cancer (SCLC) that may select patients who are likely to respond to molecularly targeted therapies. In this study, we investigate the feasibility of undertaking focused molecular analyses on routine diagnostic biopsies in patients with SCLC.

Methods: A series of histopathologically confirmed formalin-fixed, paraffin-embedded SCLC specimens were analysed for epidermal growth factor receptors (EGFR), KRAS, NRAS and BRAF mutations, ALK gene rearrangements and MET amplification. EGFR and KRAS mutation testing was evaluated using real time polymerase chain reaction (RT-PCR cobas®), BRAF and NRAS mutations using multiplex PCR and capillary electrophoresis-single strand conformation analysis, and ALK and MET aberrations with fluorescent in situ hybridization. All genetic aberrations detected were validated independently.

Results: A total of 105 patients diagnosed with SCLC between July 1990 and September 2006 were included. 60 (57 %) patients had suitable tumour tissue for molecular testing. 25 patients were successfully evaluated for all six pre-defined molecular aberrations. Eleven patients failed all molecular analysis. No mutations in EGFR, KRAS and NRAS were detected, and no ALK gene rearrangements or MET gene amplifications were identified. A V600E substitution in BRAF was detected in a Caucasian male smoker diagnosed with SCLC with squamoid and glandular features.

Conclusion: The paucity of patients with sufficient tumour tissue, quality of DNA extracted and low frequency of aberrations detected indicate that alternative molecular characterisation approaches are necessary, such as the use of circulating plasma DNA in patients with SCLC.

Poly (A)(+) Transcriptome Assessment of ERBB2-Induced Alterations in Breast Cell Lines

We report the first quantitative and qualitative analysis of the poly (A)(+) transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor) an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in ERBB2 over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as LOX, ATP5L, GALNT3, and MME revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different ERBB2 backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by ERBB2 overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.

A Randomized, Double-Blind, Placebo-Controlled Phase II Study of Vandetanib Plus Docetaxel/Prednisolone in Patients with Hormone-Refractory Prostate Cancer

Vandetanib (ZACTIMA(TM)) is a once-daily oral anticancer drug that selectively inhibits vascular endothelial growth factor receptor, epidermal growth factor receptor, and rearranged during transfection signaling. This randomized (1: 1), double-blind study evaluated vandetanib (100mg/day) or placebo in combination with docetaxel (D; 75mg/m(2) every 3 weeks) and prednisolone (P; 2 x 5 mg/day) in 86 patients with metastatic hormone-refractory prostate cancer (mHRPC). The primary assessment was prostate-specific antigen (PSA) response (confirmed reduction of >= 50% from baseline) and a greater number of patients showed a PSA response with placebo + DP (67%) versus vandetanib + DP (40%); hazard ratio = 2.23 (one-sided 80% confidence limit = 2.90; one-sided p = 0.99). More patients experienced progression events (disease progression or death from any cause) with vandetanib + DP (65%) versus placebo + DP (60%); hazard ratio = 1.13 (one-sided 80% confidence limit = 1.44; one-sided p = 0.67). The overall incidence of adverse events was similar in both groups, although more patients experienced adverse events, leading to permanent discontinuation with vandetanib + DP (28%) versus placebo + DP (12%). However, the safety and tolerability profile for vandetanib was similar to that previously reported; adverse events that occurred more frequently in the vandetanib + DP arm were hypertension (14% vs. 2%), erythematous rash (14% vs. 2%), and exfoliative rash (12% vs. 2%). In this study of patients with mHRPC, vandetanib + DP did not demonstrate any efficacy benefit, compared with placebo + DP.

Role of Fos-related antigen 1 in the progression and prognosis of ductal breast carcinoma

Aims: Fos-related antigen 1 (Fra-1) is a member of the activator protein 1 (AP-1) transcription factor family. Our objective was to evaluate the role of Fra-1 expression in breast carcinoma progression and prognosis. Methods and results: Fra-1 expression was investigated by immunohistochemistry in two tissue microarrays containing, respectively, 85 ductal carcinoma in situ (DCIS) and 771 invasive ductal carcinoma (IDC) samples. Staining was observed in the nucleus and cytoplasm of the carcinomas, but only nuclear staining was considered to be positive. Fibroblasts associated with IDC were also Fra-1-positive. The frequency of Fra-1 positivity in IDC (22.8%) was lower than that in DCIS (42.2%). No association was found between Fra-1 and clinico-pathological variables in DCIS. In IDC, Fra-1 expression correlated with aggressive phenotype markers, including: high grade, oestrogen receptor negativity and human epidermal growth factor receptor 2 (HER-2) positivity (P = 0.001, 0.015 and 0.004, respectively), and marginally with the presence of metastasis (P = 0.07). Fra-1 was more frequently positive in basal-like (34%) and in HER-2-positive (38.5%) subtypes than in luminal subtypes. Fra-1 presence did not correlate with survival. Conclusions: A high frequency of Fra-1 in DCIS tumours may be associated with early events in breast carcinogenesis. Although Fra-1 expression correlated with features of a more aggressive phenotype in IDC, no relationship with overall survival was found.

Treatment with trastuzumab for 1 year after adjuvant chemotherapy in patients with HER2-positive early breast cancer: a 4-year follow-up of a randomised controlled trial

Background Treatment with adjuvant trastuzumab for 1 year improves disease-free survival and overall survival in patients with human epidermal growth factor receptor 2 (HER2)-positive early breast cancer. We aimed to assess disease-free survival and overall survival after a median follow-up of 4 years for patients enrolled on the Herceptin Adjuvant (HERA) trial. Methods The HERA trial is an international, multicentre, randomised, open-label, phase 3 trial comparing treatment with trastuzumab for 1 and 2 years with observation after standard neoadjuvant, adjuvant chemotherapy, or both in patients with HER2-positive early breast cancer. The primary endpoint was disease-free survival. After a positive first interim analysis at a median follow-up of 1 year for the comparison of treatment with trastuzumab for 1 year with observation, event-free patients in the observation group were allowed to cross over to receive trastuzumab. We report trial outcomes for the 1-year trastuzumab and observation groups at a median follow-up of 48.4 months (IQR 42.0-56.5) and assess the effect of the extensive crossover to trastuzumab. Our analysis was by intention-to-treat. The HERA trial is registered with the European Clinical Trials Database, number 2005-002385-11. Findings The HERA trial population comprised 1698 patients randomly assigned to the observation group and 1703 to the 1-year trastuzumab group. Intention-to-treat analysis of disease-free survival showed a significant benefit in favour of patients in the 1-year trastuzumab group (4-year disease-free survival 78.6%) compared with the observation group (4-year disease-free survival 72.2%; hazard ratio [HR] 0.76; 95% CI 0.66-0.87; p<0.0001). Intention-to-treat analysis of overall survival showed no significant difference in the risk of death (4-year overall survival 89.3% vs 87.7%, respectively; HR 0.85; 95% CI 0.70-1.04; p=0.11). Overall, 885 patients (52%) of the 1698 patients in the observation group crossed over to receive trastuzumab, and began treatment at median 22.8 months (range 4.5-52.7) from randomisation. In a non-randomised comparison, patients in the selective-crossover cohort had fewer disease-free survival events than patients remaining in the observation group (adjusted HR 0.68; 95% CI 0.51-0.90; p=0.0077). Higher incidences of grade 3-4 and fatal adverse events were noted on 1-year trastuzumab than in the observation group. The most common grade 3 or 4 adverse events, each in less than 1% of patients, were congestive cardiac failure, hypertension, arthralgia, back pain, central-line infection, hot flush, headache, and diarrhoea. Interpretation Treatment with adjuvant trastuzumab for 1 year after chemotherapy is associated with significant clinical benefit at 4-year median follow-up. The substantial selective crossover of patients in the observation group to trastuzumab was associated with improved outcomes for this cohort.

PHASE I/II STUDY OF ERLOTINIB COMBINED WITH CISPLATIN AND RADIOTHERAPY IN PATIENTS WITH LOCALLY ADVANCED SQUAMOUS CELL CARCINOMA OF THE HEAD AND NECK

Purpose: Erlotinib, an oral tyrosine kinase inhibitor, is active against head-and-neck squamous cell carcinoma (HNSCC) and possibly has a synergistic interaction with chemotherapy and radiotherapy. We investigated the safety and efficacy of erlotinib added to cisplatin and radiotherapy in locally advanced HNSCC. Methods and Materials: In this Phase I/II trial 100 mg/m(2) of cisplatin was administered on Days 8, 29, and 50, and radiotherapy at 70 Gy was started on Day 8. During Phase I, the erlotinib dose was escalated (50 mg, 100 mg, and 150 mg) in consecutive cohorts of 3 patients, starting on Day 1 and continuing during radiotherapy. Dose-limiting toxicity was defined as any Grade 4 event requiring radiotherapy interruptions. Phase 11 was initiated 8 weeks after the last Phase I enrollment. Results: The study accrued 9 patients in Phase I and 28 in Phase II; all were evaluable for efficacy and safety. No dose-limiting toxicity occurred in Phase I, and the recommended Phase 11 dose was 150 mg. The most frequent nonhematologic toxicities were nausea/vomiting, dysphagia, stomatitis, xerostomia and in-field dermatitis, acneiform rash, and diarrhea. Of the 31 patients receiving a 150-mg daily dose of erlotinib, 23 (74%; 95% confidence interval, 56.8%-86.3%) had a complete response, 3 were disease free after salvage surgery, 4 had inoperable residual disease, and 1 died of sepsis during treatment. With a median 37 months` follow-up, the 3-year progression-free and overall survival rates were 61% and 72%, respectively. Conclusions: This combination appears safe, has encouraging activity, and deserves further studies in locally advanced HNSCC. (C) 2010 Elsevier Inc.

Blepharitis and trichomegaly induced by cetuximab

This report describes the case of a 41-year old woman in treatment for advanced colorectal cancer who developed severe bilateral blepharitis and trichomegaly after the second dose of cetuximab. Cetuximab-related eyelid toxicity has been described previously; however, its pathogenesis has not yet been clearly established.

Gastrointestinal stromal tumor in Brazil: Clinicopathology, immunohistochemistry, and molecular genetics of 513 cases

The aim of the present study was to evaluate the clinicopathological, immunohistochemical, and molecular genetic features of gastrointestinal stromal tumors in Brazil and compare them with cases from other countries. Five hundred and thirteen cases were retrospectively analyzed. HE-stained sections and clinical information were reviewed and the immunohistochemical expression of CD117, CD34, smooth-muscle actin, S-100 protein, desmin, CD44v3 adhesion molecule, p53 protein, epidermal growth factor receptor, and Ki-67 antigen was studied using tissue microarrays. Mutation analysis of KIT and platelet-derived growth factor receptor-alpha genes was also performed. There was a slight female predominance (50.3%) and the median age at diagnosis was 59 years. The tumors were mainly located in the stomach (38.4%). Immunohistochemistry showed that CD117 was expressed in 95.7% of cases. Epidermal growth factor receptor expression was observed in 84.4% of tumors. p53 protein expression was found only in 2.6% of cases but all belonged to the high-risk group for aggressive behavior according to the National Institutes of Health consensus approach. No CD44v3 adhesion molecule expression was detected. KIT exon 11 mutations were the most frequent (62.2%). The present data confirm that gastrointestinal stromal tumors in Brazilian patients do not differ from tumors occurring in other countries.