Introduction 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. Methods 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. Results We demonstrate that TRF2 is exclusively nuclear with a trend towards 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. Conclusion 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.

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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.

Differentiating between adenoid cystic carcinoma (AdCC) and polymorphous adenocarcinoma (PAC) can be difficult on small biopsies and cytologic specimens. As such, further characterization of their immunophenotype may aid in distinction. Previous studies have found AdCC to be SOX10+/GATA3 variable and PAC to be GATA3 negative. SOX10 expression in PAC has, as yet, not been established. We performed GATA3 and SOX10 immunohistochemistry on whole sections of 25 cases each of AdCC and PAC (including both classic PAC and the cribriform variant) to assess whether these markers are of diagnostic utility in distinguishing between these entities. SOX10 was found to be positive in 100% of PAC and AdCC whereas GATA 3 was immunoreactive in 45% of AdCCs and 20% of PAC. While this is the first series to compare SOX10 and GATA3 staining in these two tumor types, their frequent expression and similar staining patterns render them of limited value in discriminating between these neoplasms.

Infectious pseudotumors are unusual proliferations of histiocytes in response to certain microbial organisms. Occasionally this process may involve large airways, producing a mass lesion that may cause respiratory obstruction. Infectious pseudotumors can be confused with malignancy in their radiologic appearance and clinical presentation. We present a case of an aggressive endotracheal pseudotumor associated with Rhodococcus equi infection in a patient with advanced HIV disease. Microscopically, the lesion was composed of sheets of epithelioid histiocytes with large, strongly eosinophilic intra-cytoplasmic granules and features of malakoplakia. In this report, we review the literature of these unusual lesions and compare them to cases of conventional malakoplakia involving the large airways. We also explore the pathogenetic mechanisms that may contribute to the distinctive histologic appearance of Rhodococcus-associated pseudotumors.

Background: The MRN complex is essential in conserving genetic integrity. It recruits ATM kinase to damage sites, and distinguishes and activates homologous recombinational repair of DNA double-strand breaks. Individuals with mutations in the MRE11 and NBS1 genes experience radiation sensitivity and are associated with a higher cancer rate. Previously, we showed that some genes involved in DNA repair are involved in regulating cell invasiveness. Since the MRN complex is downregulated in invasive breast cancer, we hypothesize that change in MRN complex expression may contribute to the pre-invasive to invasive progression of breast cancer. Materials and Methods: The HMT-3522 series of breast epithelial cell lines containing non-invasive S1, pre-invasive S3-C, and invasive T4-2 counterparts were used in 2D or 3D culture. Western blot and immunohistochemistry (IHC) were used to determine MRE11, RAD50, and NBS1 expression levels. Breast tumor tissues each containing benign, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) from 48 patients were used after IRB approval. Two pathologists and Ariol, an automated image acquisition and analysis system, scored IHC signal. Tissues were scored 0 to 3 (0=negative, 3=strong positive). ANOVA p-value of .01 or lower was considered significant for expression comparisons. Regression analysis was used for correlating Estrogen Receptor ER/Progesterone Receptor PR status with MRN expression patterns. MRE11, RAD50, and NBS1 were downregulated using siRNA. Protein expression levels were quantified by western blot. Boyden Chamber assay was used to measure cellular invasion through laminin-rich ECM, lrECM (Matrigel). Results: In non-invasive S1 cells, 3DlrECM culturing resulted in higher levels of MRN than in 2D. In pre-invasive S3-C and invasive T4-2 cells, 3DlrECM no longer upregulated MRN. Consequently, in 3DlrECM cultures non-invasive S1 cells had a higher expression of MRE11, RAD50 and NBS1 compared to invasive T4-2 cells. IHC on tissue sections showed that, on average, normal tissues have higher levels of MRN expression than DCIS and DCIS have higher levels of expression than IDC. Multiple MRN expression patterns were observed in subsets of patients when benign, DCIS, and IDC transitions were compared. Decreases in MRN expression correlated with ER/PR negativity: “single decrease” in benign to DCIS to IDC progression was associated significantly with ER negative status for MRE11 and RAD50, not NBS1. MRE11, RAD50, and NBS1 siRNAs downregulated their protein levels in invasive T4-2 cells, resulting in a significant increase in cell invasiveness through ECM. Discussion: First, we described a new role for ECM signaling in up-regulating the DNA repair protein complex MRN in non-tumorigenic cells, which gets misregulated in pre-invasive and invasive cells resulting in a progressive decrease in MRN levels in culture and in tissues. Reciprocally, we showed that downregulation of MRN upregulates invasion through ECM, suggesting it is a negative regulator of invasion in addition to its role as a gatekeeper of genome stability. Finally, different patterns of MRN expression changes in progression associated with ER/PR status suggest MRN levels as a candidate biomarker of patient-specific clinical outcomes. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-17.

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