Elevated TRF2 in advanced breast cancers with short telomeres.

Telomere repeat binding factor 2 (TRF2) binds directly to telomeres and preserves the structural integrity of chromosome ends. In vitro models suggest that expression of TRF2 protein increases during mammary cancer progression. However, a recent study has reported that TRF2 mRNA levels tend to be lower in clinical specimens of malignant breast tissue. Here, we conduct the first large-scale investigation to assess the levels and cellular localization of the TRF2 protein in normal, pre-malignant and malignant breast tissues. Breast tissue arrays, containing normal, ductal carcinoma in situ (DCIS) and invasive carcinoma specimens, were used to assess the expression and localization of TRF2 protein. Telomere lengths were semi-quantitatively measured using a pantelomeric peptide nucleic acid probe. A mixed effects modeling approach was used to assess the relationship between TRF2 expression and telomeric signal scores across disease states or clinical staging. We demonstrate that TRF2 is exclusively nuclear with a trend toward lower expression with increased malignancy. More case-to-case variability of TRF2 immunostaining intensity was noted amongst the invasive carcinomas than the other disease groups. Invasive carcinomas also displayed variable telomere lengths while telomeres in normal mammary epithelium were generally longer. Statistical analyses revealed that increased TRF2 immunostaining intensity in invasive carcinomas is associated with shorter telomeres and shorter telomeres correlate with a higher TNM stage. All immortalized and cancer cell lines within the array displayed strong, nuclear TRF2 expression. Our data indicate that elevated expression of TRF2 is not a frequent occurrence during the transformation of breast cancer cells in vivo, but higher levels of this telomere-binding protein may be important for protecting advanced cancer cells with critically short telomeres. Our findings also reinforce the concept that serially propagated cancer cells, although tumor-derived, may not model all types of authentic tumors especially those demonstrating genetic heterogeneity.

LIM-Only Protein 4 (LMO4) and LIM Domain Binding Protein 1 (LDB1) Promote Growth and Metastasis of Human Head and Neck Cancer (LMO4 and LDB1 in Head and Neck Cancer).

Squamous cell carcinoma of the head and neck (HNSCC) accounts for more than 300,000 deaths worldwide per year as a consequence of tumor cell invasion of adjacent structures or metastasis. LIM-only protein 4 (LMO4) and LIM-domain binding protein 1 (LDB1), two directly interacting transcriptional adaptors that have important roles in normal epithelial cell differentiation, have been associated with increased metastasis, decreased differentiation, and shortened survival in carcinoma of the breast. Here, we implicate two LDB1-binding proteins, single-stranded binding protein 2 (SSBP2) and 3 (SSBP3), in controlling LMO4 and LDB1 protein abundance in HNSCC and in regulating specific tumor cell functions in this disease. First, we found that the relative abundance of LMO4, LDB1, and the two SSBPs correlated very significantly in a panel of human HNSCC cell lines. Second, expression of these proteins in tumor primaries and lymph nodes involved by metastasis were concordant in 3 of 3 sets of tissue. Third, using a Matrigel invasion and organotypic reconstruct assay, CRISPR/Cas9-mediated deletion of LDB1 in the VU-SCC-1729 cell line, which is highly invasive of basement membrane and cellular monolayers, reduced tumor cell invasiveness and migration, as well as proliferation on tissue culture plastic. Finally, inactivation of the LDB1 gene in these cells decreased growth and vascularization of xenografted human tumor cells in vivo. These data show that LMO4, LDB1, and SSBP2 and/or SSBP3 regulate metastasis, proliferation, and angiogenesis in HNSCC and provide the first evidence that SSBPs control LMO4 and LDB1 protein abundance in a cancer context.