Jareer Kassis

PLCγ contributes to metastasis of in situ-occurring mammary and prostate tumors

Phospholipase C-γ (PLCγ) has been implicated in tumor cell motility required for invasiveness and metastasis. Diminished tumor dissemination has been demonstrated in xenograft models, but studies in naturally-occurring tumors are lacking, having been limited by the timing of the interventions. Therefore, we generated mice that express a doxycycline (DOX)-inducible dominant-negative fragment of PLCγ, PLCz; this approach avoids the in utero lethality caused by the absence of PLCγ. As we targeted two de novo-occurring carcinomas of the mammary (MMTV-driven polyoma middle T antigen model, PyVmT) and prostate (TRAMP model) glands, we limited expression to these epithelial cells by driving DOX transactivator from the prostatein C3 promoter. This avoids the confounding variable of potentially abrogating motility in stromal and endothelial cells. These mice developed normally in the presence of DOX, except for limited mammary development if treated before 6 weeks and immaturity of the prostate gland if treated before 2 weeks of age. DOX-mediated induction of PLCz from age 8 to 16 weeks in PyVmT mice decreased the number of lung metastases by >10-fold (P<0.06) without a detectable effect on in situ tumor cell proliferation or tumor size. Lung metastases were also significantly decreased in the TRAMP model in which the mice expressed the PLCz fragment (P<0.05). DOX treatment itself had no effect on tumor size or metastasis in control mice, nor did it affect tumor dissemination in nontransgenic littermates. In conclusion, abrogation of the PLCγ signaling pathway can limit the metastatic potential of carcinomas.

3D in vitro tissue models and their potential for drug screening

Introduction: The development of one standard, simplified in vitro three-dimensional tissue model suitable to biological and pathological investigation and drug-discovery may not yet be feasible, but standardized models for individual tissues or organs are a possibility. Tissue bioengineering, while concerned with finding methods of restoring functionality in disease, is developing technology that can be miniaturized for high throughput screening (HTS) of putative drugs. Through collaboration between biologists, physicists and engineers, cell-based assays are expanding into the realm of tissue analysis. Accordingly, three-dimensional (3D) micro-organoid systems will play an increasing role in drug testing and therapeutics over the next decade. Nevertheless, important hurdles remain before these models are fully developed for HTS. Areas covered: We highlight advances in the field of tissue bioengineering aimed at enhancing the success of drug candidates through pre-clinical optimization. We discuss models that are most amenable to high throughput screening with emphasis on detection platforms and data modeling. Expert opinion: Modeling 3D tissues to mimic in-vivo architecture remains a major challenge. As technology advances to provide novel methods of HTS analysis, so do potential pitfalls associated with such models and methods. We remain hopeful that integration of biofabrication with HTS will significantly reduce attrition rates in drug development.

EBV-expressing AGS gastric carcinoma cell sublines present increased motility and invasiveness.

Tumor invasion marks a critical point in cancer progression; it is a harbinger of morbidity and mortality. Thus, the cellular events that enable the invasive phenotype are under intense investigation. Epstein‐Barr virus (EBV) is associated with a number of cancers, including Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC) and is suspected to contribute to their tumorigenesis. On average, 8% of gastric carcinomas have been shown to carry this virus. To explore whether the presence of EBV in gastric carcinoma contributes to tumor progression in this predominantly invasive carcinoma, we examined a panel of 2 in vitro EBV‐infected human gastric cancer cell line sublines and their mock‐infected AGS parental control line. We found EBV infection caused a marked increase in transmigration of a Matrigel barrier (415% and 303%, p < 0.05, for the 2 infected lines). This correlated with increased motility of these sublines (233% and 140%, p < 0.05). As this pattern of increased motility leading to a more pronounced enhancement of invasion has been noted in other tumor cells, we explored the roles of autocrine signaling pathways previously implicated in carcinoma motility and invasion. Inhibitors to the epidermal growth factor receptor (EGFR) (PD153035), phospholipase C (PLC) (U73122), extracellular‐signal regulated kinase (ERK)/mitogen‐activated protein kinase (MAPK) (PD089035) and PI‐3 kinase (Wortmannin) were not informative. These data suggest that EBV increases migration of AGS cells by a mechanism independent of these autocrine growth factor‐induced pathways. Instead, we found that the EBV‐infected cells presented increased focal adhesion kinase (FAK) phosphorylation. These findings suggest a role for integrin‐mediated signaling in promoting EBV‐associated invasiveness.

Motility is rate-limiting for invasion of bladder carcinoma cell lines

Induced migration of tumor cells is generally considered to be one critical step in cancer progression to the invasive and metastatic stage. The implicit caveat of studies that show this is that other, unknown, signaling pathways and biophysical events are actually the operative rate-limiting steps, and not motility per se. Thus, to examine the hypothesis that motility is a single, but overall rate-limiting function required for invasion, disparate motility processes need be blocked with concordant effects on tumor invasion. Recently, we and others have described two signaling pathways that are critical to growth factor-induced motility but not mitogenesis. The key molecular switches are phospholipase C-gamma (PLCgamma) and calpain for cytoskeletal reorganization and rear detachment, respectively. We examined this hypothesis in a highly invasive tumor, bladder carcinoma. Three different human tumor cell lines, 253J-B-V, UMUC and T-24, were tested for invasiveness in vitro by transmigration of a Matrigel barrier. Inhibiting PLCgamma with the pharmacologic agent U73122 or the molecular dominant-negative PLCz construct reduced both invasiveness and motility. The same was noted when calpain was blocked using calpain inhibitor I (ALLN). These results demonstrate that one interventional target for limiting invasion is not necessarily an individual motility pathway but rather cell migration per se.

Dolichol-phosphate-mannose-3 (DPM3)/prostin-1 is a novel phospholipase C-γ regulated gene negatively associated with prostate tumor invasion

The most ominous development in tumor progression is the transition to an invasive and metastatic phenotype. Little is known, however, about the molecular alterations that cause a tumor to become invasive. In view of this, we have used microarray expression analysis to evaluate the expression profiles of a unique panel of human DU145 prostate cancer sublines that vary in their invasive potential. The three DU145 sublines expressed epidermal growth factor (EGF) receptors that differed in their ability to activate phospholipase C-γ (PLCγ). Three-way analyses yielded 11 genes out of 4608 genes screened that associated directly or inversely with invasive potential. The gene whose expression correlated most strongly with lack of invasion was identified as a potential invasion suppressor and called prostin-1. Pharmacological inhibition of PLCγ (U73122) confirmed that PLCγ signaling suppressed prostin-1 in that U73122 treatment caused induction of prostin-1 in PLCγ competent cells. The prostin-1 gene, conserved through phylogeny, is induced by androgen in LNCaP cells and encodes a 92 amino acid protein. The protein shares no extensive homologies with other known genes, yet was recently identified as a small stabilizer subunit of the dolichol-phosphate-mannose (DPM) synthase complex. That DPM3/prostin-1 might suppress tumor progression was supported by the finding that exogenous expression in COS cells leads to apoptosis. These findings support the use of model cell lines to identify putative tumor suppressors and promoters.