Tarlochan Nijjar

Loss of p53 function accelerates acquisition of telomerase activity in indefinite lifespan human mammary epithelial cell lines

We describe novel effects of p53 loss on immortal transformation, based upon comparison of immortally transformed human mammary epithelial cell (HMEC) lines lacking functional p53 with closely related p53(+) lines. Our previous studies of p53(+) immortal HMEC lines indicated that overcoming the stringent replicative senescence step associated with critically short telomeres (agonescence), produced indefinite lifespan lines that maintained growth without immediately expressing telomerase activity. These telomerase(−) ‘conditionally immortal’ HMEC underwent an additional step, termed conversion, to become fully immortal telomerase(+) lines with uniform good growth. The very gradual conversion process was associated with slow heterogeneous growth and high expression of the cyclin-dependent kinase inhibitor p57Kip2. We now show that p53 suppresses telomerase activity and is necessary for the p57 expression in early passage p53(+) conditionally immortal HMEC lines, and that p53(−/−) lines exhibit telomerase reactivation and attain full immortality much more rapidly. A p53-inhibiting genetic suppressor element introduced into early passages of a conditionally immortal telomerase(−) p53(+) HMEC line led to rapid induction of hTERT mRNA, expression of telomerase activity, loss of p57 expression, and quick attainment of uniform good growth. These studies indicate that derangements in p53 function may impact malignant progression through direct effects on the conversion process, a potentially rate-limiting step in HMEC acquisition of uniform unlimited growth potential. These studies also provide evidence that the function of p53 in suppression of telomerase activity is separable from its cell cycle checkpoint function.

Accumulation and altered localization of telomere-associated protein TRF2 in immortally transformed and tumor-derived human breast cells.

We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked upregulation (10–15 fold) of the telomere-binding protein, TRF2. Upregulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite lifespan and immortal HMEC. TRF2 protein was not upregulated by the oncogenic agents alone in the absence of immortalization, nor by expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least twofold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells’ ability to maintain an indefinite lifespan.

2A4 binds soluble and insoluble light chain aggregates from AL amyloidosis patients and promotes clearance of amyloid deposits by phagocytosis (

Abstract Amyloid light chain (AL) amyloidosis is characterized by misfolded light chain (LC) (amyloid) deposition in various peripheral organs, leading to progressive dysfunction and death. There are no regulatory agency–approved treatments for AL amyloidosis, and none of the available standard of care approaches directly targets the LC protein that constitutes the amyloid. NEOD001, currently in late-stage clinical trials, is a conformation-specific, anti-LC antibody designed to specifically target misfolded LC aggregates and promote phagocytic clearance of AL amyloid deposits. The present study demonstrated that the monoclonal antibody 2A4, the murine form of NEOD001, binds to patient-derived soluble and insoluble LC aggregates and induces phagocytic clearance of AL amyloid in vitro. 2A4 specifically labeled all 21 fresh-frozen organ samples studied, which were derived from 10 patients representing both κ and λ LC amyloidosis subtypes. 2A4 immunoreactivity largely overlapped with thioflavin T–positive labeling, and 2A4 bound both soluble and insoluble LC aggregates extracted from patient tissue. Finally, 2A4 induced macrophage engagement and phagocytic clearance of AL amyloid deposits in vitro. These findings provide further evidence that 2A4/NEOD001 can effectively clear and remove human AL-amyloid from tissue and further support the rationale for the evaluation of NEOD001 in patients with AL amyloidosis.

Abstract 355: A monoclonal antibody against laminin alpha 4 inhibits both MCAM and integrin-a6b1 binding, blocks tumor cell adhesion in vitro and slows melanoma tumor growth in vivo

Laminin is an extracellular matrix molecule that is secreted as a trimeric protein composed of an α, β and γ chain. There are five distinct laminin α chains that impart distinct binding and signaling activities for cell adhesion, migration, and differentiation in a tissue-specific fashion. Laminin a4 (LAMA4) is expressed by vascular endothelial cells throughout the body, but is also overexpressed by cells within tumors of different origin compared to healthy human tissue. LAMA4 serves as a ligand for the cell adhesion receptors MCAM and a6b1 integrin, as well as for cell surface heparin sulfate proteoglycans. In order to probe how the MCAM/Laminin interaction contributes to tumor growth, we screened for LAMA4-specific monoclonal antibodies that block MCAM binding. We identified several such antibodies, and found that a subset also inhibits LAMA4 binding to a6b1 integrin, indicating that MCAM and a6b1 integrin recognize a similar region of LAMA4. Examining cells from multiple types of tumors, we found that these antibodies were potent inhibitors of tumor cell adhesion to LAMA4 in vitro. Finally, with in vivo administration, we demonstrated dose-dependent inhibition of human melanoma tumor growth in a mouse xenograft model, accompanied by morphologic changes in LAMA4 tumor distribution. These findings suggest that the MCAM/a6b1 integrin binding activity of LAMA4 contributes to both tumor adhesion and growth and that LAMA4 may provide a therapeutic drug target for several different oncology indications. Citation Format: Stephen J. Tam, Lauri E. Li, Carlos Lorenzana, Yue Liu, Ken Flanagan, Philip J. Dolan, Lana Alexander, Josh Salmans, Robin M. Barbour, Jeffrey N. Higaki, Tarlochan Nijjar, Wagner Zago, Ted A. Yednock, Gene Kinney. A monoclonal antibody against laminin alpha 4 inhibits both MCAM and integrin-a6b1 binding, blocks tumor cell adhesion in vitro and slows melanoma tumor growth in vivo. [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 355. doi:10.1158/1538-7445.AM2015-355