| Abstract|| |
Introduction: Breast cancer is the commonest cause of death among middle aged women. BRCA1 associated tumors carry a poor prognosis. Angiogenesis is considered necessary for tumor growth and for its metastasis. Hypoxia stimulates HIF-1α which then activates transcription of various proangiogenic cytokines like VEGF. In the present study we examined HIF-1α expression, sVEGF levels and BRCA1 mutations and their relation with clinicopathological parameters. We also determined whether the angiogenic markers have different role in angiogenesis in BRCA1 related cancers as compared to sporadic breast cancers. Materials and Methods: The study was conducted on 50 cases of breast cancer specimens. Histopathological typing and grading was done followed by immunohistochemistry for BRCA1 and HIF-1α. VEGF was done in the serum by ELISA. Results: All the tumors were infiltrating ductal carcinoma NOS. 16 cases were reported grade II and 34 cases as grade III. On immunohistochemistry, 27 cases showed BRCA1 positivity and HIF-1α was positive in 39 cases. sVEGF levels were increased in 21 cases (42%). BRCA1 positivity, HIF-1α expression and increased VEGF levels were significantly associated with higher grade and lymph node metastasis. There was significant correlation of BRCA1 positivity with increased HIF-1α expression (P = 0.009) and increased sVEGF levels (P = 0.005). Conclusion: Our findings suggest that BRCA 1 positive tumors have unique molecular profile and different mechanism of tumorigenesis. Such tumors are associated with increased HIF-1α expression and VEGF levels.
Keywords: BRCA1, breast cancer, hypoxia- induced factor-1, immunohistochemistry, vascular endothelial growth factor
|How to cite this article:|
Sharma M, Piplani S, Madan M, Manjari M, Garg S, Kaur K. Hypoxia-induced factor-1 alpha, vascular endothelial growth factor expression in BRCA1-related breast cancer: A prospective study in tertiary care hospital. Indian J Pathol Microbiol 2017;60:469-74
|How to cite this URL:|
Sharma M, Piplani S, Madan M, Manjari M, Garg S, Kaur K. Hypoxia-induced factor-1 alpha, vascular endothelial growth factor expression in BRCA1-related breast cancer: A prospective study in tertiary care hospital. Indian J Pathol Microbiol [serial online] 2017 [cited 2018 Jan 19];60:469-74. Available from: http://www.ijpmonline.org/text.asp?2017/60/4/469/222982
| Introduction|| |
Breast cancer is the most common malignancy in women and is the most common cause of death in developed countries in middle-aged women. It is becoming frequent in developing countries as well. It is estimated that by 2020, the number of breast cancer cases will exceed cases of cervical cancer in India and also it occurs decade earlier among Indian women.
Mutations in tumor suppressor gene BRCA1 are responsible in about 40%–50% of hereditary breast cancers and most of the hereditary breast-ovarian syndrome. Women with an abnormal BRCA1 gene have up to 85% risk of developing breast cancer by the age of 70. BRCA1-associated tumors are genetically different from sporadic cases and tend to occur in younger women and have higher grade and lack hormonal receptors, so they are associated with poor prognosis. BRCA1 mutation is followed by p53 dysfunction and hormonal receptors negativity, suggesting some intriguing mechanism of interaction between BRCA1 and other molecular markers.
Angiogenesis is considered necessary for tumor growth and its metastasis as well. It is a highly regulated process, in which a balance of pro- and anti-angiogenic molecules is maintained and the stage at which angiogenesis occurs in tumor progression in known as angiogenic switch.
Vascular endothelial growth factor (VEGF) is considered vital factor playing a role in angiogenesis. VEGF family consists of five different polypeptides and VEGF-A being the predominant and best-studied angiogenic factor. Relative lack of oxygen stimulates hypoxia-induced factor 1 alpha (HIF-1α), an oxygen-sensitive transcription factor which then activates transcription of various proangiogenic cytokines such as VEGF. Hypoxia occurs in the tumor environment due to increased consumption of the oxygen due to the rapid proliferation of the cells outgrowing the existing vascularity. HIF consists of two subunits: HIF α and HIF β. Both the subunits are constitutively expressed, but under normoxic conditions, prolyl hydroxylase domain (PHD)-containing enzymes modify α subunit causing polyubiquitinated α subunit. Such polyubiquitinated α subunits are quickly degraded by cellular proteasome apparatus. However, in hypoxia, low oxygen tension limits the activity of PHD enzymes, thus leaving α subunit undehydroxylated resulting in increase of HIF α levels and activity. This HIF-1 complex binds with hypoxia-responsive elements in the promoter region of target genes including VEGF, thus increasing its expression, resulting in angiogenesis.
In the present study, we examined HIF-1α expression, soluble VEGF (sVEGF) levels, and BRCA1 mutations and their relation with clinicopathological parameters. We, also, determined whether the angiogenic markers have different role in angiogenesis in BRCA1-related cancers as compared to sporadic breast cancer cases.
| Materials and Methods|| |
The study was conducted on 50 cases of breast cancer received as lumpectomy or mastectomy. Detailed clinical data were recorded as per the pro forma attached. Cases with positive family history and history of contralateral breast cancer and ovarian cancers (cases which were more likely to be associated with BRCA1 mutation) were included in the study.
The tissue was formalin-fixed and paraffin-embedded and then stained for hematoxylin and eosin for histopathological typing and grading [Figure 1] and [Figure 2]. All the cases were subjected to immunohistochemistry for BRCA1 and HIF-1α expression. VEGF was done in the serum samples which collected by enzyme-linked immunosorbent assay (ELISA).
Immunohistochemistry was performed using antibodies against BRCA1 (Biocare medical) and HIF-1α (Diagnostic Biosystem). The antigen retrieval was done using a pressure cooker method with 10-mmol citrate buffer at pH 6.0. Tris buffer was used as wash buffer. Endogenous activity was blocked using hydrogen peroxide. After protein blocking, slides were incubated overnight with anti-BRCA1 and HIF-1α antibodies and were conjugated with streptavidin-horseradish peroxidase. Diaminobenzidine tetrahydrochloride was used as the chromogen. The slides were counterstained with hematoxylin and were examined by light microscopy. More than 30% nuclei-stained brown were taken positive for BRCA1 [Figure 3] and [Figure 4]. For HIF-1α, brown nuclei and cytoplasmic staining were taken positive [Figure 5] and [Figure 6].
|Figure 5: Hypoxia-induced factor-1 alpha positivity (nuclear and cytoplasmic strong intensity) (immunohistochemistry, ×400)|
Click here to view
|Figure 6: Hypoxia-induced factor-1 alpha positivity (nuclear strong intensity) (immunohistochemistry, ×400)|
Click here to view
- 1 = Nuclear staining <1% of cells
- 2 = Nuclear staining 1%–10% of cells and/or with weak cytoplasmic staining
- 3 = Nuclear staining 10%–50% of cells and/or with distinct cytoplasmic staining
- 4 = Nuclear staining >50% of cells and/or with distinct cytoplasmic staining.
For VEGF, serum sample of every patient was processed using ELISA kit (Ray Biotech, Inc.). The step solution changed the color from blue to yellow, and the intensity of the color was measured at 450 nm. Color developed was proportionate to the levels of bound VEGF. The minimum detectable level was <10 pg/ml. The normal range was taken 45–280 pg/ml.
| Results|| |
The age of the patients varied from 26 to 70 years, with the maximum number of the patients (54%) belonging to age group of 41–60 years. Right side was slightly more commonly involved and upper outer quadrant being most commonly involved site (68%). The size varied from 1.5 to 5 cm with the maximum number of cases being in group of 2–5 cm (72%). All the tumors were infiltrating ductal carcinoma not otherwise specified. Sixteen cases were reported Grade II and 34 cases as Grade III. There was no case in Grade I. Lymph nodes were recovered in 44 cases of radical mastectomy. Out of which, 26 cases showed metastatic carcinomatous deposits.
On immunohistochemistry, 27 cases showed BRCA1 positivity, and HIF-1α was positive in 39 cases. Serum VEGF levels were increased in 21 cases (42%). BRCA1 positivity was observed in 65% of cases in Grade III tumors as compared to 35% of cases in Grade II tumors (P = 0.004). Similarly, lymph node involvement was significantly higher in BRCA1-positive cases, and BRCA1 positivity was associated with the increase in size of tumor [Table 1].
|Table 1: Correlation of BRCA1 with grading of tumor, lymph node status, and size of the tumor|
Click here to view
On correlating grade of the tumor with HIF-1α score, the score tends to be greater in higher grade, though no significant correlation was found. Similarly, HIF-1α higher scores were associated with increase in lymph nodes involvement [Table 2].
|Table 2: Correlation of hypoxia-induced factor 1 alpha with grading of tumor and lymph node status|
Click here to view
Similarly, increased sVEGF levels were seen in 14 cases of Grade III as compared to seven cases of Grade II. Increased sVEGF was significantly correlated with the metastasis in the lymph node with P = 0.001. 17/21 cases were positive for metastasis in the lymph node as compared to 9/29 cases with normal sVEGF which had secondary deposits in the lymph node.
On correlating the VEGF levels with the HIF-1α score, 19/21 (90%) cases showing increased VEGF levels showed positive HIF-1α expression, thus proving that VEGF levels are positively correlated with HIF-1α expression.
On correlating BRCA1 positivity with HIF-1α expression and VEGF levels, it was found that 25/27 (92.5%) of BRCA1-positive cases were also positive for HIF-1α as compared to 14/23 (60.8%) of BRCA1-negative cases which showed HIF-1α expression (P = 0.009). Therefore, BRCA1 positivity was significantly correlated with the expression of HIF-1α. 16 out of 27 (59%) BRCA1-positive cases had increased VEGF levels as compared to 5 out of 23 (22%) BRCA1-negative cases which had increased sVEGF levels, thus correlating BRCA1 positivity with increased sVEGF significantly (P = 0.005) [Table 3].
|Table 3: Correlation of BRCA1 status with hypoxia-induced factor 1 alpha expression and vascular endothelial growth factor levels|
Click here to view
| Discussion|| |
The aggressive biological behavior of breast cancer is mostly the result of changes in molecular characteristics of tumor cells, including alterations in mechanisms controlling adhesion, growth, and proliferation. A wide variety of morphology-based and molecular-based breast cancer prognostic factors and tumor markers had been studied to identify the oncogenes involved in initiation and progression of tumors and development of new anticancer drugs. The incidence of breast cancer in India is on the increase and is becoming number one cancer in females in metropolitan cities.
Earlier, BRCA1 and BRCA2 were estimated to be responsible for 75% of familial breast cancers. Recent data show this percentage to vary depending on population studied. Immunohistochemical expression of BRCA1 protein has been studied as inexpensive and valuable preliminary method for detecting BRCA1 status with high sensitivity and specificity of anti-BRCA1 antibodies. BRCA1 positivity indicates the full-length functional protein, while negativity may be seen in sporadic BRCA1 mutation., In the present study, 27 cases showed immunopositivity for BRCA1. Of 27 BRCA1-positive cases, 18 (67%) cases were of Grade III tumors as compared to 9 (33%) cases of Grade II tumors, thus significantly correlating the BRCA1 expression with higher grade of tumor (P = 0.004). Similar results have been seen in various previous studies which have shown that BRCA1-related cancers were of higher grade., BRCA1-positive cases had higher number of lymph node involvement and tumor size >2 cm at the time of presentation. These results are in concordance with results of various researches.,
HIF-1α was positive in 39/50 (78%) of the cases correlating the findings in other studies where their percentage varied from 69 to 76%., While correlating the grade of tumor with HIF-1α, it was found that higher scores of HIF-1α had seen in Grade III cases as compared to Grade II cases, though no significant correlation was calculated. HIF-1α expression increases with higher grade of the tumor which has been concluded in the previous studies as well., Out of 26 cases positive for secondary carcinomatous deposits in lymph node, 21 had increased expression of HIF-1α. These results were similar to those reported previously in the literature.,
Increased VEGF levels were seen in 14 cases of Grade III as compared to seven cases of Grade II. 17/21 cases were positive for metastasis in the lymph nodes as compared to 9/29 cases with normal VEGF levels which had secondary carcinomatous deposits in the lymph node. Increased VEGF levels were significantly correlated with the metastasis in lymph nodes (P = 0.001). Several other studies have evaluated the positive correlation of increased VEGF levels with grade and lymph node metastasis.,, On correlating the VEGF levels with HIF-1α score, 19/21 cases showing increased VEGF levels which showed positive HIF-1α expression, thus proving that VEGF levels are positively correlated with HIF-1α expression. These results were consistent with previous reports.,
Correlating the BRCA1 positivity with HIF-1α expression and increased sVEGF levels, it was concluded that there was significant correlation of BRCA1 positivity with increased HIF-1α expression as 25/27 (92.5%) BRCA1-positive cases were also positive for HIF-1α as compared to 14/23 (60.8%) BRCA1-negative cases, which showed HIF-1α expression with significant P = 0.009. Similarly, BRCA1-positive cases had significant increased VEGF levels (P = 0.005), with 16/27 (59%) BRCA1 cases showing increased sVEGF levels as compared to 5/23 (22%) BRCA1-negative cases which had increased VEGF levels. Similar results had been observed by Yan et al. and Saponaro et al. in their studies., While some other researchers have found the positive correlation between BRCA1 and HIF-1α expression but lower VEGF levels in BRCA1 mutation cases.
Thus, BRCA1 positivity, HIF-1 α expression, and VEGF level were associated with poor clinicopathological factors such as higher grade of the tumor, greater size of the tumor, and lymph node metastasis.
There was positive significant correlation between BRCA1 positivity and increased VEGF levels, thus proving that BRCA1 mutation is associated with increased angiogenesis through the expression of HIF-1α and VEGF. Such association had been demonstrated in vitro study where BRCA1 protein blocked VEGF promoter activity by estrogen receptor alpha explaining the increased VEGF levels in BRCA1 carrier. BRCA1-positive tumors enhance the hypoxic drive and promote hypoxia-driven ER degradation due to suppressed PHD-containing enzymes. These suppressed PHD enzymes result in increase in HIF-1α levels and its activity, thus leading to increased VEGF levels. Aggressive behavior of BRCA1 mutation can be explained by molecular level interaction between BRCA1 protein and angiogenic factor (HIF-1 α and VEGF), causing new capillary formation responsible for tumor growth and metastasis as well.
| Conclusion|| |
Our study concludes that BRCA1-positive tumors are associated with increased HIF-1α expression and increased VEGF levels as compared to BRCA1-negative tumors. The angiogenesis resulted due to expression of HIF-1α and VEGF plays an important role in the aggressive behavior of BRCA1 mutation cancers. Further studies should determine the role of novel combination therapy of DNA-damaging agents plus antiangiogenic drugs in BRCA1-positive cases to improve therapeutic opportunities in such patients.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Calys-Tagoe BN, Yarney J, Kenu E, Amanhyia NA, Enchill E, Obeng I, et al.
Profile of cancer patients' seen at Korle Bu teaching hospital in Ghana (a cancer registry review). BMC Res Notes 2014;7:577.
Shetty P. India faces growing breast cancer epidemic. Lancet 2012;379:992-3.
Kama IM, Gabal AK, El-Naggar S, Shaaban HY, Shehata M. BRCA1 gene expression in breast cancer in relation to other prognostic markers in Egyptian women. Anatol J Obstet Gynecol 2011;1:1-5.
Comănescu M, Popescu CF. BRCA1 expression in invasive breast carcinomas and clinicopathological correlations. Rom J Morphol Embryol 2009;50:419-24.
Kim S, Rimm D, Carter D, Khan A, Parisot N, Franco MA, et al.
BRCA status, molecular markers, and clinical variables in early, conservatively managed breast cancer. Breast J 2003;9:167-74.
Sensi E, Tancredi M, Aretini P, Cipollini G, Naccarato AG, Viacava P, et al.
P53 inactivation is a rare event in familial breast tumors negative for BRCA1 and BRCA2 mutations. Breast Cancer Res Treat 2003;82:1-9.
Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996;86:353-64.
Nowak DG, Woolard J, Amin EM, Konopatskaya O, Saleem MA, Churchill AJ, et al.
Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors. J Cell Sci 2008;121:3487-95.
Stricker TP, Kumar V. Neoplasia. In: Kumar V, Abbas AK, Fausto N, Aster JC, editors. Robbins and Cotran Pathologic Basis of Disease. 8th
ed. Philadelphia: Saunders; 2010. p. 297-8.
Barreto de Melo RM, Beltrao EI. Prognostic and predictive biomarkers for hypoxic regions on breast cancer: Advances and challenges. J Bras Patol Med Lab 2013;491:67-70.
Bruick RK, McKnight SL. A conserved family of prolyl-4-hydroxylases that modify HIF. Science 2001;294:1337-40.
King MC, Levy-Lahad E, Lahad A. Population-based screening for BRCA1 and BRCA2: 2014 Lasker award. JAMA 2014;312:1091-2.
Al-Mulla F, Abdulrahman M, Varadharaj G, Akhter N, Anim JT. BRCA1 gene expression in breast cancer: A correlative study between real-time RT-PCR and immunohistochemistry. J Histochem Cytochem 2005;53:621-9.
Burkadze G, Khardzeishvili O, Gudadze M, Tsikhiseli G, Turashvili G. Immunohistochemical expression of BRCA1 protein in invasive ductal carcinoma of the breast. Georgian Med News 2010;184:51-60.
Lakhani SR, Gusterson BA, Jacquemier J, Sloane JP, Anderson TJ, van de Vijver MJ, et al.
The pathology of familial breast cancer: Histological features of cancers in families not attributable to mutations in BRCA1 or BRCA2. Clin Cancer Res 2000;6:782-9.
Palacios J, Honrado E, Osorio A, Cazorla A, Sarrió D, Barroso A, et al.
Immunohistochemical characteristics defined by tissue microarray of hereditary breast cancer not attributable to BRCA1 or BRCA2 mutations: Differences from breast carcinomas arising in BRCA1 and BRCA2 mutation carriers. Clin Cancer Res 2003;9:3606-14.
Atchley DP, Albarracin CT, Lopez A, Valero V, Amos CI, Gonzalez-Angulo AM, et al.
Clinical and pathologic characteristics of patients with BRCA-positive and BRCA-negative breast cancer. J Clin Oncol 2008;26:4282-8.
Kallergi G, Markomanolaki H, Giannoukaraki V, Papadaki MA, Strati A, Lianidou ES, et al.
Hypoxia-inducible factor-1alpha and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients. Breast Cancer Res 2009;11:R84.
Ni X, Zhao Y, Ma J, Xia T, Liu X, Ding Q, et al.
Hypoxia-induced factor-1 alpha upregulates vascular endothelial growth factor C to promote lymphangiogenesis and angiogenesis in breast cancer patients. J Biomed Res 2013;27:478-85.
Bos R, Zhong H, Hanrahan CF, Mommers EC, Semenza GL, Pinedo HM, et al.
Levels of hypoxia-inducible factor-1 alpha during breast carcinogenesis. J Natl Cancer Inst 2001;93:309-14.
Gruber G, Greiner RH, Hlushchuk R, Aebersold DM, Altermatt HJ, Berclaz G, et al.
Hypoxia-inducible factor 1 alpha in high-risk breast cancer: An independent prognostic parameter? Breast Cancer Res 2004;6:R191-8.
Zhang J, Yin L, Wu J, Zhang Y, Xu T, Ma R, et al.
Detection of serum VEGF and MMP-9 levels by luminex multiplexed assays in patients with breast infiltrative ductal carcinoma. Exp Ther Med 2014;8:175-80.
Ali EM, Sheta M, El Mohsen MA. Elevated serum and tissue VEGF associated with pooroutcome in breast cancer patients. Alexandria J Med 2011;47:217-24.
Shan X, Wang D, Chen J, Xiao X, Jiang Y, Wang Y, et al.
Necrosis degree displayed in computed tomography images correlated with hypoxia and angiogenesis in breast cancer. J Comput Assist Tomogr 2013;37:22-8.
Yan M, Rayoo M, Takano EA, KConFab Investigators, Fox SB. BRCA1 tumours correlate with a HIF-1alpha phenotype and have a poor prognosis through modulation of hydroxylase enzyme profile expression. Br J Cancer 2009;101:1168-74.
Saponaro C, Malfettone A, Ranieri G, Danza K, Simone G, Paradiso A, et al.
VEGF, HIF-1α expression and MVD as an angiogenic network in familial breast cancer. PLoS One 2013;8:e53070.
Kang HJ, Kim HJ, Rih JK, Mattson TL, Kim KW, Cho CH, et al.
BRCA1 plays a role in the hypoxic response by regulating HIF-1alpha stability and by modulating vascular endothelial growth factor expression. J Biol Chem 2006;281:13047-56.
Kawai H, Li H, Chun P, Avraham S, Avraham HK. Direct interaction between BRCA1 and the estrogen receptor regulates vascular endothelial growth factor (VEGF) transcription and secretion in breast cancer cells. Oncogene 2002;21:7730-9.
Dr. Manisha Sharma
B-241, Ranjit Avenue, Amritsar, Punjab
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]