Summary Alterations in the p53 gene were analysed in 39 patients with locally advanced breast cancers (LABCs) (stage III-IV) with inflammatory signs in most cases (UICC stage T4d = 32 patients) by molecular and immunohistochemical (IHC) approaches. All patients were included in the same therapy protocol. Using polymerase chain reaction (PCR) and a single-strand conformational polymorphism migration technique (SSCP), the presence of mutations in exons 2-11, covering the entire coding sequence of the p53 gene, was evaluated. Using the mouse specific anti-human p53 monoclonal antibody (PAb 1801), we also looked for overexpression of the p53 protein in tissue sections. In 16 cases shifted bands were reproducibly identified by PCR-SSCP, and all but one (localised to exon 10) were in exons 5-8, the usual mutational hotspots. Fifteen of these 16 samples were sequenced and 14 of the suspected mutations (36%) were confirmed. Most of them (12) were single nucleotide substitutions, and transitions were more frequent (eight cases) than transversions (four cases). Fourteen of the tumour samples were positively stained with the monoclonal antibody PAb 1801, 11 with nuclear staining only, two with mixed cytoplasmic and nuclear staining and one with cytoplasmic staining only. Staining patterns were very heterogeneous in terms of the percentage of positive cells (10-75%) and their distribution in the tissue section (isolated foci or dispersed cells). In 11 of the 14 mutated cases a positive immunostaining was observed. The presence of a p53 mutation was significantly associated with larger tumour diameter (X2 = 7.490, P = 0.0062) and the presence of clinical metastases (stage IV) (X2 = 10.113, P = 0.0015). A non-statistically significant trend of association was observed between p53 mutation, negative oestrogen receptors and lower response rate to therapy. Our results in this group of patients and the heterogeneity of the staining of tumour cells in tissue sections suggest that p53 mutations could be a late event in this non-familial form of breast cancer.

The mRNA levels of the ras-related human rhoA, rhoB and rhoC genes were studied in human breast-cancer cell lines (HBCal), and in normal and immortalized mammary epithelial cells (HMEC) by Northern blot analysis and in situ hybridization. In contrast to the ubiquitous rhoA and rhoC gene expression, dramatic variations in the mRNA level of the rhoB gene were evidenced. The rhoB mRNA level appeared to be inversely correlated to the amounts of the epidermal-growth-factor(EGF) receptors in these cells. The rhoB transcripts were detected at high levels in ZR75-1, MCF7, HSL 53, HSL 59, HSL 90, T47D and SKBR3 HBCal, at hardly detectable levels in BT 20, MDA-MB 231 and H466B HBCal and at intermediate levels in normal and immortalized breast epithelial cells. Rapid and transient induction of the rhoB transcription was observed after EGF treatment in serum-deprived MDA-MB231, T47D and immortalized epithelial cells. In contrast, no modulation of rhoB expression by EGF could be objectified in the MCF7 and ZR75-1 cell lines. Yet a normal function of EGF receptors was evidenced, since the immediate early gene c-fos was rapidly induced, suggesting a constitutive expression of rhoB in these cell lines bypassing the regulation by EGF. In human mammary epithelial cells, rhoB mRNA is rapidly and transiently induced with EGF concentrations known to stimulate cell proliferation. This suggests that the rhoB product might be involved in a cascade that initiates or promotes cell proliferation, and plays an important role in EGF-stimulated growth of breast normal and cancer cells.

Formation of new blood (angiogenesis) and lymphatic (lymphangiogenesis) vessels are major events associated with most epithelial malignancies, including breast cancer. Angiogenesis is essential for cancer cell survival. Lymphangiogenesis is critical in maintaining tumoral interstitial fluid balance and importing tumor-facilitatory immune cells. Both vascular routes also serve as conduits for cancer metastasis. Intratumoral hypoxia promotes both events by stimulating multiple angiogenic/lymphangiogenic growth factors. Studies on tumor-associated lymphangiogenesis and its exploitation for therapy have received less attention from the research community than those on angiogenesis. Inflammation is a key mediator of both processes, hijacked by many cancers by aberrant expression of the inflammation-associated enzyme cyclo-oxygenase (COX)-2. In this review, we focus on breast cancer and show that COX-2 is a major promoter of both events, primarily resulting from the activation of Prostaglandin (PG) E receptor EP4 on tumor cells, tumor-infiltrating immune cells, and endothelial cells; and induction of oncogenic microRNAs. COX-2/EP4 pathway also promotes additional events in breast cancer progression, such as cancer cell migration, invasion, and stimulation of stem–like cells. Based on a combination of studies using multiple breast cancer models, we show that EP4 antagonists hold a major promise in breast cancer therapy in combination with other modalities including immune check-point inhibitors

No abstract