Creation of Formalin-Fixed, Paraffin-Embedded 3D Lung Cancer Cellular Spheroids for the Optimization of Immunohistochemistry Staining Procedures.

In an era of precision medicine important treatment decisions are dictated by expression of clinically informative tumor protein biomarkers. These biomarkers can be detected by immunohistochemistry (IHC) performed in tumor tissue sections obtained from biopsies or resections. Like all experimental procedures, IHC needs optimization for several of its steps. However, the investigator must avoid optimizing the IHC procedure using valuable human biopsy samples which may be difficult to obtain. Ideally, valuable biopsy samples should only be subjected to IHC once the IHC protocol has been optimized. In this chapter, we describe a procedure for IHC optimization using tri-dimensional (3D) cellular spheroids created from cultured cells. In this approach, cultured cells are pelleted into 3D spheroids, which are then processed just like a tissue sample, namely, fixed, embedded, sectioned, mounted on slides, and stained with IHC just like a human tissue sample. These 3D cellular spheroids have a tissue-like architecture and cellularity resembling a tumor section, and both cellular and antigen structure are preserved. This method is therefore acceptable for IHC optimization before proceeding to the IHC staining of human tumor samples.

Immunoblot Validation of Phospho-Specific Antibodies Using Lung Cancer Cell Lines.

The cancer phenotype is usually characterized by deregulated activity of a variety of cellular kinases, with consequent abnormal hyper-phosphorylation of their target proteins. Therefore, antibodies that allow the detection of phosphorylated versions of proteins have become important tools both preclinically in molecular cancer research, and at the clinical level by serving as tools in pathological analyses of tumors. In order to ensure reliable results, validation of the phospho-specificity of these antibodies is extremely important, since this ensures that they are indeed able to discriminate between the phosphorylated and unphosphorylated versions of the protein of interest, specifically recognizing the phosphorylated variant. A recommended validation approach consists in dephosphorylating the target protein and assessing if such dephosphorylation abrogates antigen immunoreactivity when using the phospho-specific antibody. In this chapter, we describe a protocol to validate the specificity of a phospho-specific antibody that recognizes a phosphorylated variant of the Retinoblastoma (Rb) protein in lung cancer cell lines. The protocol consists in the dephosphorylation of the Rb-containing protein lysates by treating them with bovine intestinal phosphatase, followed by assessment of the dephosphorylation by immunoblot.

Characterization of epidermal growth factor receptor (EGFR) P848L, an unusual EGFR variant present in lung cancer patients, in a murine Ba/F3 model.

Lung cancer patients with mutations in epidermal growth factor receptor (EGFR) benefit from treatments targeting tyrosine kinase inhibitors (TKIs). However, both intrinsic and acquired resistance of tumors to TKIs are common, and EGFR variants have been identified that are resistant to multiple TKIs. In the present study, we characterized selected EGFR variants previously observed in lung cancer patients and expressed in a murine bone marrow pro-B Ba/F3 cell model. Among these EGFR variants, we report that an exon 20 deletion/insertion mutation S768insVGH is resistant to erlotinib (a first-generation TKI), but sensitive to osimertinib (a third-generation TKI). We also characterized a rare exon 21 germline variant, EGFR P848L, which transformed Ba/F3 cells and conferred resistance to multiple EGFR-targeting TKIs. Our analysis revealed that P848L (a) does not bind erlotinib; (b) is turned over less rapidly than L858R (a common tumor-derived EGFR mutation); (c) is not autophosphorylated at Tyr 1045 [the major docking site for Cbl proto-oncogene (c-Cbl) binding]; and (d) does not bind c-Cbl. Using viability assays including 300 clinically relevant targeted compounds, we observed that Ba/F3 cells transduced with EGFR P848L, S768insVGH, or L858R have very different drug-sensitivity profiles. In particular, EGFR P848L, but not L858R or S768insVGH, was sensitive to multiple Janus kinase 1/2 inhibitors. In contrast, cells driven by L858R, but not by P848L, were sensitive to multikinase MAPK/extracellular-signal-regulated kinase (ERK) kinase and ERK inhibitors including EGFR-specific TKIs. These observations suggest that continued investigation of rare TKI-resistant EGFR variants is warranted to identify optimal treatments for cancer.

Detection of Retinoblastoma Protein Phosphorylation by Immunoblot Analysis.

The retinoblastoma tumor suppressor protein (pRb) is a preeminent tumor suppressor that acts as a cell cycle repressor, specifically as an inhibitor of the G1-S transition of the cell cycle . pRb is a phosphoprotein whose function is repressed by extensive phosphorylation in several key residues, and therefore, pRb's phosphorylation status has become a surrogate for pRb activity. In particular, hyperphosphorylation of pRb has been associated with pathological states such as cancer, and therefore, assessing pRb's phosphorylation status is increasingly gaining diagnostic and prognostic value, may be used to inform therapeutic decisions, and is also an important tool for the cancer biologists seeking an understanding of the molecular etiology of cancer. In this chapter, we discuss an immunoblot protocol to detect pRb phosphorylation in two residues, serine 612 and threonine 821, in protein extracts from cancer cells.

Immunohistochemical Detection of Retinoblastoma Protein Phosphorylation in Human Tumor Samples.

The retinoblastoma protein (pRb) is an important tumor suppressor and cell cycle repressor. pRb is a phosphoprotein whose function is regulated primarily at the level of phosphorylation, and therefore, detecting pRb's phosphorylation status in human tissue samples can be clinically informative. Unfortunately, detection of phosphorylated pRb residues can be technically challenging, as these residues can often be weak antigens. In this chapter, we describe an enhanced sensitivity immunohistochemistry protocol for the staining of phosphorylated serine 249 in pRb, in human lung tumor samples.

Hyper-phosphorylation of Rb S249 together with CDK5R2/p39 overexpression are associated with impaired cell adhesion and epithelial-to-mesenchymal transition: Implications as a potential lung cancer grading and staging biomarker.

Prediction of lung cancer metastasis relies on post-resection assessment of tumor histology, which is a severe limitation since only a minority of lung cancer patients are diagnosed with resectable disease. Therefore, characterization of metastasis-predicting biomarkers in pre-resection small biopsy specimens is urgently needed. Here we report a biomarker consisting of the phosphorylation of the retinoblastoma protein (Rb) on serine 249 combined with elevated p39 expression. This biomarker correlates with epithelial-to-mesenchymal transition traits in non-small cell lung carcinoma (NSCLC) cells. Immunohistochemistry staining of NSCLC tumor microarrays showed that strong phospho-Rb S249 staining positively correlated with tumor grade specifically in the squamous cell carcinoma (SCC) subtype. Strong immunoreactivity for p39 positively correlated with tumor stage, lymph node invasion, and distant metastases, also in SCC. Linear regression analyses showed that the combined scoring for phospho-Rb S249, p39 and E-cadherin in SCC is even more accurate at predicting tumor staging, relative to each score individually. We propose that combined immunohistochemistry staining of NSCLC samples for Rb phosphorylation on S249, p39, and E-cadherin protein expression could aid in the assessment of tumor staging and metastatic potential when tested in small primary tumor biopsies. The intense staining for phospho-Rb S249 that we observed in high grade SCC could also aid in the precise sub-classification of poorly differentiated SCCs.

Somatic Mutations and Ancestry Markers in Hispanic Lung Cancer Patients.

INTRODUCTION: To address the lack of genomic data from Hispanic/Latino (H/L) patients with lung cancer, the Latino Lung Cancer Registry was established to collect patient data and biospecimens from H/L patients. METHODS: This retrospective observational study examined lung cancer tumor samples from 163 H/L patients, and tumor-derived DNA was subjected to targeted-exome sequencing (>1000 genes, including EGFR, KRAS, serine/threonine kinase 11 gene [STK11], and tumor protein p53 gene [TP53]) and ancestry analysis. Mutation frequencies in this H/L cohort were compared with those in a similar cohort of non-Hispanic white (NHW) patients and correlated with ancestry, sex, smoking status, and tumor histologic type. RESULTS: Of the adenocarcinomas in the H/L cohort (n = 120), 31% had EGFR mutations, versus 17% in the NHW control group (p < 0.001). KRAS (20% versus 38% [p = 0.002]) and STK11 (8% versus 16% [p = 0.065]) mutations occurred at lower frequency, and mutations in TP53 occurred at similar frequency (46% versus 40% [p = 0.355]) in H/L and NHW patients, respectively. Within the Hispanic cohort, ancestry influenced the rate of TP53 mutations (p = 0.009) and may have influenced the rate of EGFR, KRAS, and STK11 mutations. CONCLUSIONS: Driver mutations in H/L patients with lung adenocarcinoma differ in frequency from those in NHW patients associated with their indigenous American ancestry. The spectrum of driver mutations needs to be further assessed in the H/L population.

The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts.

We previously characterized the retinoblastoma tumor suppressor protein (Rb) as a regulator of adherens junction assembly and cell-to-cell adhesion in osteoblasts. This is a novel function since Rb is predominantly known as a cell cycle repressor. Herein, we characterized the molecular mechanisms by which Rb performs this function, hypothesizing that Rb controls the activity of known regulators of adherens junction assembly. We found that Rb represses the expression of the p21-activated protein kinase (Pak1), an effector of the small Rho GTPase Rac1. Rac1 is a well-known regulator of adherens junction assembly whose increased activity in cancer is linked to perturbations of intercellular adhesion. Using nuclear run-on and luciferase reporter transcription assays, we found that Pak1 repression by Rb is transcriptional, without affecting Pak1 mRNA and protein stability. Pak1 promoter bioinformatics showed multiple E2F1 binding sites within 155 base pairs of the transcriptional start site, and a Pak1-promoter region containing these E2F sites is susceptible to transcriptional inhibition by Rb. Chromatin immunoprecipitations showed that an Rb-E2F complex binds to the region of the Pak1 promoter containing the E2F1 binding sites, suggesting that Pak1 is an E2F target and that the repressive effect of Rb on Pak1 involves blocking the trans-activating capacity of E2F. A bioinformatics analysis showed elevated Pak1 expression in several solid tumors relative to adjacent normal tissue, with both Pak1 and E2F increased relative to normal tissue in breast cancer, supporting a cancer etiology for Pak1 up-regulation. Therefore, we propose that by repressing Pak1 expression, Rb prevents Rac1 hyperactivity usually associated with cancer and related to cytoskeletal derangements that disrupt cell adhesion, consequently enhancing cancer cell migratory capacity. This de-regulation of cell adhesion due to Rb loss could be part of the molecular events associated with cancer progression and metastasis.

Lipopolysaccharides induce intestinal serum amyloid A expression in the sea cucumber Holothuria glaberrima.

We have previously characterized the first invertebrate homolog of serum amyloid A (SAA) proteins in the sea cucumber Holothuria glaberrima, where its expression is associated with intestinal regeneration, suggesting a possible involvement of SAA proteins in intestinal morphogenesis. Here we show that bacterial lipopolysaccharides (LPS) trigger a coelomocyte-mediated immune response in H. glaberrima, inducing an approximately threefold increase in coelomocyte phagocytic activity. Furthermore, LPS induces an approximately fourfold increase in SAA mRNA levels in non-regenerating intestines. These results show that in H. glaberrima, LPS act as an immune activator and that SAA expression can be modulated by immune-associated processes.

Building a long distance training program to enhance clinical cancer research capacity in Puerto Rico.

Barriers persist in the development and delivery of effective cancer therapies to under-represented minority populations. In Puerto Rico, cancer is the second leading cause of death, yet cancer research awareness and training opportunities remain somewhat limited on the island. These limitations hinder progress toward decreasing the cancer health disparities that exist within the Puerto Rican population. The predominantly Hispanic population of Puerto Rico is the focus of a partnership between the Ponce Health Sciences University-Medical School and Ponce Research Institute (PHSU) in Ponce, Puerto Rico and the H. Lee Moffitt Cancer Center in Tampa, Florida. The Partnership goals are to reduce these barriers through an integrated, multipronged approach of training and education alongside outreach and research components. This report describes the approaches, successes and challenges of enhancing clinical cancer research capacity on the island and the unique challenges of a partnership between two institutes physically separated by long distances. Once fully developed this model may be exportable to other Latin American countries where the need is even greater.

A role for the retinoblastoma protein as a regulator of mouse osteoblast cell adhesion: implications for osteogenesis and osteosarcoma formation.

The retinoblastoma protein (pRb) is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis.