Audrey A. Rasmussen

Insulin-like growth factor mediated stromal-epithelial interactions in human breast cancer

The prominent ‘desmoplastic’ or stromal reaction seen in many invasive breast carcinomas lead to early speculation that stromal cells play a role in breast cancer pathogenesis [1]. Experimental evidence now supports this hypothesis and interactions between stromal cells and epithelial cells appear to be important for both normal mammary development and neoplasia. The identification of genes that are selectively expressed in the stroma of malignant breast lesions has recently provided new insights into the molecular basis of stromal-epithelial interactions. Stromally expressed genes include growth factors, proteases and extracellular matrix proteins, all biological activities with potential roles in malignant progression. Investigations discussed here concern the nature of the paracrine signals provided by malignant epithelial cells that activate changes in stromal gene expression, the effect that the stromally derived factors have on the behavior of malignant epithelial cells and the identification of novel factors and receptors in either stroma or epithelia that contribute to their mutual interactions. These questions will be addressed in the context of this laboratorys studies on insulin-like growth factors, as these molecules show marked differences in stromal expression between benign and malignant breast tissue and thus provide a useful paradigm for investigations into the paracrine environment of an evolving breast tumor.

Decreased cisplatin/DNA adduct formation is associated with cisplatin resistance in human head and neck cancer cell lines

Purpose: To evaluate the correlation between cisplatin sensitivity, intracellular glutathione, and platinum/DNA adduct formation (measured by atomic absorption spectroscopy) in a series of seven head and neck cancer cell lines, and to evaluate the effect of biochemical modulation of glutathione on platinum/DNA adduct formation and repair. Methods: Cisplatin/DNA adducts were measured by atomic absorption spectroscopy. Glutathione content was measured by enzymatic assay and was modulated with buthionine sulfoximine. Apoptosis was measured by double-labeled flow cytometry. Results: Intracellular glutathione concentration was strongly correlated with cisplatin resistance (P = 0.002, R2=0.7). There was also a statistically significant inverse correlation between cisplatin/DNA adduct formation and the IC50 for cisplatin in these cell lines. (P=0.0004, R2=0.67). In addition, resistant cells were able to repair approximately 70% of cisplatin/DNA adducts at 24 h, while sensitive cells repaired less than 28% of adducts in the same period. However, despite the positive correlation between cellular glutathione and cisplatin resistance, there was no direct correlation between intracellular glutathione concentration and platinum/DNA adduct formation. Further, depletion of intracellular glutathione by buthionine sulfoximine did not dramatically alter formation of cisplatin/DNA adducts even though it resulted in marked increase in cisplatin cytotoxicity and was associated with increased apoptosis. Conclusions: These results suggest that glutathione has multiple effects not directly related to formation of cisplatin/DNA adducts, but may also be an important determinant of the cells ability to repair cisplatin-induced DNA damage and resist apoptosis.

Prognostic value of c-erbB2 and other markers in patients treated with chemotherapy for recurrent head and neck cancer

Chemotherapy is widely used in patients with recurrent head and neck cancer, but no clear markers are available that can predict response to treatment or survival in these patients.

Glutathione content but not gamma glutamyl cysteine synthetase mRNA expression predicts cisplatin resistance in head and neck cancer cell lines

Abstract Purpose: To correlate cellular glutathione content and γ-glutamyl cysteine synthetase (γGCS) mRNA expression with cisplatin sensitivity in a panel of seven head and neck squamous cancer cell lines. Methods: Cisplatin IC50 was determined for each cell line using a sodium tetreazolium (XTT) assay. Cellular glutathione content was measured by using a previously reported enzymic method. γGCS mRNA expression was measured using an RNase protection assay. Results: Total cellular glutathione was an excellent predictor of cisplatin sensitivity in this series of cell lines. The IC50 for cisplatin in the cell line with the highest glutathione concentration was approximately 90 times higher than in the cell line with the lowest glutathione concentration. Regression analysis showed a highly statistically significant positive correlation between cisplatin IC50 and cellular glutathione (coefficient of determination R2=0.81, P=0.0012). Somewhat surprisingly, in contrast to previous studies in ovarian cancer, γGCS mRNA expression in these cell lines was not significantly predictive of either total cellular glutathione or cisplatin sensitivity (R2=0.005, P=0.84). As expected, treatment of resistant cell lines with buthionine sulfoximine resulted in decreased cellular glutathione and enhanced cisplatin sensitivity. Conclusions: Our results suggest that glutathione may be an important determinant of cisplatin sensitivity in clinical head and neck cancer. Since cisplatin is the most active chemotherapy drug for the treatment of this disease, this correlation may have important clinical relevance. The lack of correlation between glutathione level and γGCS expression suggests that salvage or alternate synthetic pathways may be critical in these cells.

Stromal IGF-II messenger RNA in breast cancer: Relationship with progesterone receptor expressed by malignant epithelial cells

In breast cancer, insulin-like growth factor II (IGF-II) is stromal in origin and is considered an important regulator of tumour epithelium growth. The presence of progesterone receptor (PR) is expression of an intact oestrogen regulatory pathway of breast malignant epithelial cells and represents a parameter of cell differentiation in breast cancer. In this study we have examined the relationship between IGF-II mRNA expression and ER, PR content in 75 breast cancer. Formalin-fixed paraffin-embedded tissue sections were used to preserve histological details. IGF-II mRNA was evaluated by in situ hybridisation method and ER, PR by immunohistochemistry. IGF-II mRNA was scored semi-quantitatively: 2.6% breast tumour specimen expressed no IGF-II mRNA, 46.7% had low levels of expression (IGF-II -) and 50.7% had moderate or high IGF-II mRNA content (IGF-II +). IGF-II mRNA was found in the stroma fibroblasts surrounding malignant lesions and no signal was detected in malignant epithelial cells. In contrast, ER and PR were expressed only by neoplastic epithelial cells and no immunoreactivity was found in the stroma: 50/75 (66.6%) breast cancer specimens were positive for ER (ER+) and 35 (46.6%) for PR (PR+). Both, IGF-II mRNA and PR were directly correlated with the stromal proliferation (p<0.05 and p<0.001, respectively). No relationship was found between IGF-II RNA and ER. In contrast 24/35 (73.5%) PR breast cancer tissues were IGF-II+(p<0.01) and a strong correlation was found between epithelial PR immunostaining and stromal IGF-II mRNA content (p<0.003). Our data indicate that in breast cancer IGF-II mRNA is generally expressed by stromal cells and ER and PR by ephitelial cancer cells, and that IGF-II mRNA expression is strongly related with both percentage and staining intensity of PR+ epithelial cancer cells. These data support the hypothesis that IGF-II produced by the fibroblasts may exert a paracrin effect on malignant epithelium regulating its differentiation.

Insulin-like growth factor II (IGF-II) immunohistochemistry in breast cancer: relationship with the most important morphological and biochemical prognostic parameters.

Recent in situ hybridization experiments have shown a high content of IGF-II mRNA in breast cancer stroma. The aim of this study was to examine the relationship between IGF-II protein expression and several prognostic parameters in 75 infiltrating ductal carcinomas (IDC) of the breast. Tissue sections were evaluated for proliferative activity, IGF-II protein, ER, PgR, p53, and p21 expression using immunohistochemical procedures. The degree of stromal proliferation was assessed. Menopausal status, axillary lymph node involvement and nuclear grade were known. Thirty-five patients (44.3%) were premenopausal and 47 (62.6%) had lymph node metastases. Marked stromal proliferation was found in 34 (45.3%) specimens and high nuclear grade in 20 (26.5%). Eighteen tumors (24%) showed no IGF-II immunostaining. In the positive cases, IGF-II was detected both in the tumor stroma and in the cytoplasm of epithelial cancer cells: a high IGF-II content was found in 12 specimens (16.0%), a low content in 14 (18.7%) and a moderate content in 31 (41.3%). Twenty-four tumors (32.0%) showed high proliferative activity. Both ER and PgR were expressed in the nucleus of cancer cells: 49 tumors (65.3%) were ER positive (ER+) and 34 (45.3%) PgR positive (PgR+). p21 protein was detected in 37 tumors (49.6%) and p53 in 12 (16%). IGF-II protein was not correlated with menopausal status, lymph node metastases, nuclear grade, proliferative activity, ER or p53. In contrast, IGF-II correlated strongly with stromal proliferation (p=0.008), PgR (p=0.03) and p21 (p=0.01). This study demonstrates that in IDC of the breast IGF-II protein is expressed in the epithelium and stroma of the majority of tumors and is correlated with stromal amount, PgR and p21 expression. These preliminary results indicate that IGF-II expression in breast cancer is connected with two important regulators of breast cancer growth and differentiation.

Interactions Between Stroma and Epithelium in Breast Cancer

Breast cancers are traditionally considered epithelial malignancies. In reality, breast cancers, like normal breast itself, are complex tissues with many interdependent components. Although it is the epithelial cell from ductal or lobular elements within the breast which undergoes malignant transformation, no breast cancer can grow beyond microscopic size and become clinically significant without a complex and dynamic interaction with the surrounding stroma. Stromal elements, consisting of matrix proteins, fibroblasts, vascular elements, and their myriad cellular products, are critical determinants of the growth and malignant behavior of a breast cancer. Ongoing growth and dissemination of a breast malignancy requires complex, but poorly understood, interactions between these elements.