LÉO RAMOSFor more than a century, doctors and researchers have paid overriding attention to tumors in diagnosing, treating and understanding the origin and possible development of dozens of forms of cancer. Tumor cells are still the main actors on the cancer stage, but for some years the scope of interest has been expanding and now attention is turning as well to the supporting role that normal cells play in advancing tumor development, which may provide clues to why tumors evolve differently or why treatments work in different ways in different people.
In São Paulo, teams at two institutions – the School of Medicine of the University of São Paulo (USP) and A.C. Camargo Hospital – have ascertained that the support cells in many tissues, known as fibroblasts, can be found in the tumor microenvironment and can produce factors that promote tumor growth. “The tumor microenvironment can be decisive to tumor growth,” says Maria Aparecida Koike Folgueira, researcher at USP. The microenvironment consists of stromal cells, mainly fibroblasts, and of extracellular matrix components, which are found inside or around the tumor. In one experiment, USP researchers Maria Mitzi Brentani and Folgueira evaluated the gene expression profile by culturing cancer cells and fibroblasts from a breast tumor and a lymph node (a center for producing defense cells) compromised by the disease. They observed a reciprocal influence between the cells, which prompted a change in the gene expression of both the fibroblasts and the cancer cells. They also verified that the fibroblasts increase the spread and invasiveness of breast cancer cells. The São Paulo teams are now working to sequence messenger RNA in fibroblasts to uncover changes that may occur in such microenvironment cells and that might be involved in prognosis of the disease. In collaboration with Barretos Cancer Hospital, they are also assessing the influence of stromal cells on the response to chemotherapy.
In another line of study, they are investigating why breast cancer, a disease that appears mainly in older women, may in certain cases present in young women. Since one of the causes of cancer is an accumulation of genetic mutations that leads to uncontrolled cell proliferation, the disease is normally linked to aging, which in turn is linked to the accumulation of defects in DNA molecules.
Called tumor suppressors, the BRCA1 and BRCA2 genes take part in DNA repair. When these genes are defective, they account for up to 10% of breast tumors and 15% of ovarian tumors. Another gene, known as TP53 – the so-called guardian of the genome – monitors cell proliferation and interrupts it when it detects something abnormal, at least until the DNA repair system can move into action and make the needed corrections (see illustration). Mutations in this gene can disrupt its functioning and give rise to various types of cancer.
“The factors that influence early-onset breast cancer are still not well understood,” observes Folgueira. “We’re examining mutations and the molecular profile of the tumor to see whether there’s any feature that distinguishes it and helps explain why it appears earlier.” Dirce Carraro and Maria Isabel Achatz, of A.C. Camargo Hospital, and Folgueira and Brentani, of USP, are evaluating mutations in the BRCA1, BRCA2 and TP53 genes of women who had breast cancer between the ages of 20 and 35. They have observed that BRCA1 and BRCA2 mutations are more often found in young patients with a reported family history of cancer, but that mutation of TP53 is not common. In an extension of the study, conducted in conjunction with the São Paulo State Cancer Institute (Icesp), Folgueira is trying to identify whether habits, environmental factors and somatic mutations (observed only in the tumor and not hereditary) may be other factors implicated in the development of cancer in young women.
Another arm of their research is an effort to evaluate the effects of hormones on breast cancer. The sex hormones estrogen and progesterone serve as factors in promoting breast cancer by stimulating the proliferation of breast cells. Another hormone, vitamin D, which is produced in the skin through reactions between 7-dehydrocholesterol and UV-B rays, may have an antiproliferative effect. However, this effect has only been demonstrated in in vitro studies on cell lines exposed to high concentrations of the hormone, which can have undesirable side effects in vivo, such as hypercalcemia. In view of this evidence, the USP group, in partnership with the Brazilian Institute for Cancer Control (IBCC), is studying how proliferation and gene expression of the tumor can be impacted by the action of calcitriol – an active form of vitamin D – at concentrations not associated with side effects.
Hormones and vitamins
After ascertaining that breast cancer patients tend towards vitamin D insufficiency or deficiency, the researchers analyzed the effect of supplementing with calcitriol for a short time (one month) prior to surgery in women with breast cancer who had already entered menopause. The disease did not advance during this period, that is, the tumors did not grow. However, in analyzing the tumors collected before and after supplementation, researchers did note that vitamin D has a very subtle impact on gene expression. Through studies of animal xenografts (tumor implants), the group is now evaluating whether intratumoral injection of vitamin D can have an antiproliferative effect.
One of the group’s central efforts is the search for markers of chemotherapy response. The São Paulo teams believe they can use genetic markers to predict the efficacy of the chemotherapy. The hunt for molecular markers of therapy response has been facilitated by DNA chips – also called microarrays – that enable analysis of the expression of many genes simultaneously.
In 2005, using the DNA chips developed by the Ludwig Cancer Research Institute, Brentani and Folgueira tested 4,608 genes at one time in RNA samples extracted from breast tumors from fifty-one women between the ages of 51 and 67, looking for differences that could tell them something new about the evolution of the disease. In the midst of all their data, they found a set of three genes – PRSS11, CLPTM1 and MTSS1 – that could identify which patients would be responsive to chemotherapy and which patients would not, one of the big problems faced by oncologists. Many tumors look perfectly treatable in tests.
One of the strategies is to start with chemotherapy to reduce the size of the mass to be surgically removed and then go on to the operation itself. However, not all patients respond the same way. While the tumor mass will be reduced in most women who received doxorubicin – the key component in the chemotherapy cocktail – about 20% apparently do not respond to the procedure. What’s even more crucial: there’s no way to know beforehand who will benefit from the treatment.
Two years ago, Brentani and Folgueira showed that it is possible to replace microarrays with a cheaper technique, called PCR (polymerase chain reaction), which amplifies the signal of genes activated by the multiplication of DNA molecules. However, in this study they used fresh frozen tumors.
At hospitals, on the other hand, cancer samples are put in paraffin and then sent for testing. The next step is therefore to show that the same results can be obtained when RNA is extracted from paraffin-embedded material. If successful, this will represent a medical application of knowledge produced in the laboratory to more accurately detect and treat a disease with complex features and specificities.
1. Molecular markers of response to neoadjuvant chemotherapy in breast cancer (nº 2001/00146-8) (2001-2004); Grant mechanism Regular line of assistance for research project; Coordinator Maria Mitzi Brentani – USP School of Medicine; Investments R$515,088.21
2. Gene expression in fibroblasts associated with breast carcinomas classified in sub-types according to estrogen and progesterone receptors and C-ERBB2 (nº 2009/10088-7) (2009-2012); Grant mechanism Thematic project; Investments R$1,492,318.72.
3. Heterotypic signaling between epithelial tumor cells and fibroblasts in breast carcinoma (nº 2004/04607-8) (2005-2008); Grant mechanism Thematic project; Coordinator Maria Mitzi Brentani – USP School of Medicine; Investments R$755,501.06.
BARROS FILHO, M. C. et al. Gene trio signatures as molecular markers to predict response to doxorubicin cyclophosphamide neoadjuvant chemotherapy in breast cancer patients. Braz J Med Biol Res. v.43, n. 12, p. 1225-31, 2010.
FOLGUEIRA, M. A. et al. Gene expression profile associated with response to doxorubicin-based therapy in breast cancer. Clin Cancer Res. v. 11, n. 20, p. 7434-43, 2005.
ROZENCHAN, P.B. et al. Reciprocal changes in gene expression profiles of co-cultured breast epithelial cells and primary fibroblasts. Int J Cancer. v. 125, n. 12, p. 2767-77, 2009.
From our archives
The first signs of alert – Issue 115 – September 2005
Gene mutation may define breast cancer prognosis – Issue 26 – November 1997