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A CROSS-SECTIONAL STUDY OF RANKL AND NF-ΚΒ LEVELS AMONG POSTMENOPAUSAL BREAST CANCER PATIENTS ATTENDING AHMADU BELLO UNIVERSITY TEACHING HOSPITAL, ZARIA NIGERIA: PRELIMINARY INVESTIGATIONS AND IMPLICATIONS ON DISEASE SUBTYPES, SEVERITY AND THERAPY

 

 

Abubakar Abdullahi Maru1, *Aliyu Muhammad1, Sanusi Bello Mada1, Abur Peter Pase2, 

Saad Aliyu Ahmed3, Sani Ibrahim1, Saadatu Ibrahim1, Gilead Ebiegberi Forcados4, 

Uche Samuel Ndidi1, Murtala Abubakar3

 

1Department of Biochemistry, Faculty of Life Sciences, Ahmadu Bello University, Zaria, Kaduna State, Nigeria; 2Department of Surgery, Ahmadu Bello University Teaching Hospital Zaria; 3Department of Pathology, Ahmadu Bello University Teaching Hospital Zaria; 4Biochemistry Division, 

National Veterinary Research Institute, Vom. Nigeria

 

ABSTRACT

 

Background: Breast cancer incidence and mortality is characterized by variations in disease pathogenesis and treatment outcomes across racial and ethnic boundaries in which RANKL and NF-κβ levels are central especially among postmenopausal breast cancer patients. Aims and Objectives: This study sought to examine if RANKL, NF-κβ and oxidative stress levels were associated with disease subtypes, severity, and chemotherapy courses among the postmenopausal breast cancer patients attending Ahmadu Bello University Teaching Hospital. Materials and Methods: We examined RANKL and NF-κβ levels in serum of postmenopausal breast cancer patients attending Ahmadu Bello University Teaching Hospital (ABUTH) using ELISA kits. Sociodemographic characteristic and clinical parameters of the patients were documented using questionnaires. Results: The mean age of the respondents was 54.7 ± 6.7 years, while the modal age was 45-50 years. Triple negative breast cancer subtype accounted for 40% of the patients, while 83.3% had invasive carcinoma histological type. Serum RANKL was significantly (P<0.05) higher (40%) in breast cancer patients when compared to apparently healthy control, but lowest in triple negative patients when compared to other subtypes. NF-κβ concentration was significantly (P<0.05) higher (83.7%) in breast cancer patients but highest among triple negative and HER2-enriched patients when compared to apparently healthy. NF-κβ correlated with third chemotherapy course of chemotherapy. Oxidative stress markers were significantly (P<0.05) higher in breast cancer patients when compared to apparently healthy control. A significant (P<0.05) association between RANKL, NF-κβ concentrations and disease severity (stage I to stage IV) was also observed.  Conclusion: RANKL and NF-κβ levels were associated with disease subtypes, severity, and chemotherapy courses among the postmenopausal breast cancer patients. This could serve as a preliminary source of information for further studies on their prognostic value.

 

Keywords: Breast cancer; NF-kB; RANKL; Sociodemographic parameters; Oxidative stress


INTRODUCTION


Breast cancer is a type of cancer that develops in the breast cells [1]. Typically, the cancer forms either in the lobules, the ducts, the fatty tissue, or the fibrous connective tissue within the breast. It is now a commonly diagnosed disease even in developing countries with an incidence rate of 50.5 per 100,000 in Nigeria, implying that 1 out of every 2000 women in Nigeria stands the risk of being diagnosed with breast cancer each year [2]. Reports show that there is an increasing rate of mortality among breast cancer patients in Sub-Saharan Africa, and Nigeria contributes greatly to the recorded mortality cases in the region  [3]. Accumulating evidence from molecular examination suggests that breast cancers with different histopathological and biological features exhibit varying levels of disease aggressiveness and response to treatment [4.] Breast cancer is a heterogeneous condition consisting of multiple subtypes with distinct morphologies [5]. Breast cancer is subdivided into different groups based on their origin. Luminal A and B originate from the mammary duct luminal epithelium with consistent hormone receptor expression [5] The luminal A tumors have higher expression of estrogen receptor related genes and lower expression of proliferative genes (ER+, PR+, HER2-) when compared to luminal B cancers (ER+, PR+, HER2-) [6]. Tumors classified as HER2 overexpressing are a group of aggressive breast cancers that are associated with poor prognosis, expressed only HER2 and termed as HER2-enriched [7]. Basal like tumors are tumors that originate from mammary basal myo-epithelium and are classified as group of aggressive breast cancers that are characterized by negative expression of hormone receptors (ER-, PR-, and HER2-) thus termed Triple negative breast cancer [8]. Triple negative breast cancer follows an aggressive clinical course with difficulty of standard targeted systemic therapy[8]. It is characterized by rapid growth and is commonly seen among African women and women of African descent [9]. The tumors are reported to be larger than other subtypes and metastasis among patients is seen to have a tendency towards extending to visceral organs [8]. 

A number of breast cancer predisposing factors have been identified, prominent among which are age, early menarche, genetics, breast feeding, exogenous hormone exposure, cigarette smoking, alcohol use and obesity[10]. However, these predisposing factors can vary based on individuals, ethnicity, and geographical location [11]. Breast cancer is more prevalent among post-menopausal women, as women aged 60 – 70 years have the highest risk of developing breast cancer [12]. An increased risk for developing breast cancer is also associated with the number of first-degree relatives of an individual who have been diagnosed with breast cancer[13]. Early menarche and late menopause have also been identified as a risk factor for breast cancer, due to longer duration of circulating estrogen in this category of women [14]. Women with no history of breast feeding are also at risk of developing breast cancer because they usually have continuous circles of estrogen synthesis  [15].

As a signaling intermediary biomolecule, RANKL has also been implicated in mammary cell proliferation, breast cancer initiation and metastasis to bone [16]. Receptor Activator of Nuclear factor Kappa beta Ligand (RANKL) is an important molecule that plays crucial role not only in bone metabolism and osteoporosis 17, but also in the development of the mammary gland during pregnancy [18]. Downstream of RANKL, Nuclear Factor kappa Beta (NF-kB) is a transcription factor that controls physiological functions that are observably altered during breast cancer[19]. NF-kB contributes to breast cancer via mechanisms involving mammary cell proliferation, inflammation, resistance to apoptosis and metastasis [20]. Altered NF-kB expression has been associated with larger tumor size and aggressive disease progression in a number of breast cancer patients [21,22]. Research carried out in our laboratories have previously examined biochemical alterations associated with breast carcinogenesis in in vivo models [23,24] and the ameliorative potential of phytonutrients[25–28]. However, studies are needed to further understand biochemical alterations related to inflammatory processes, response to therapy and their possible association with socio-demographic factors among postmenopausal breast cancer patients in the sub-region. This study was carried out at the Ahmadu Bello University Teaching Hospital (ABUTH) which is the largest teaching hospital in North-Western Nigeria, the largest geopolitical zone in Nigeria with a population of about 39,000,000 people [29].  

MATERIALS AND METHODS

Study Population

This cross-sectional study was carried out at the surgical outpatient clinic of Ahmadu Bello University Teaching Hospital (ABUTH) Zaria between June 2019 and December 2020. The respondents were breast cancer patients aged 46 – 80 years. A convenient sampling technique was used for the study. Validated semi-structured questionnaires were employed in assessing the socio-demographic characteristics of the patients. Sixty (60) breast cancer patients and thirty (30) apparently healthy individuals were used for the study. Ethical approval (ABUTHZ/HREC/W41/2020) was obtained from the health research and ethics Committee of the ABUTH, Zaria, in accordance with the Helsinki Declaration, and a voluntary written informed consent was obtained from each participant before recruitment into the study. 

All female breast cancer patients diagnosed with bone disorders, cardiovascular diseases, HIV, or other forms of cancers and patients that are alcoholics and cigarette smokers were excluded. Patients with induced menopause and those aged 46 years but still menstruating were also excluded from the study. The selected breast cancer patients were categorized into four (luminal A, luminal B, HER2-enriched, triple negative) based on their receptor status as determined by immunohistochemical studies.

Blood Sample Collection

About 5ml of blood sample from each participating patient was collected separately into sterilized dry plain tubes and allowed to clot for about 30 minutes. It was then centrifuged at 3500 rpm for 15 minutes and the serum obtained was stored in the refrigerator at -20° until further analysis.  

Measurement of Serum RANKL

RANKL concentration was determined in serum samples using ELISA Kit (Wuhan Fine Biotech Co., Ltd, China, Batch No. H0313F033). According to Manufacturer’s instruction. Briefly all reagents and samples were allowed to equilibrate to room temperature (18-260C) and then mixed well. Exactly 100µl of the prepared standard was aliquot into the standard wells while 100µl of sample dilution buffer was added into the blank well and 100µl of the sample was then properly added into the test sample wells. The plate was sealed with a cover and incubated at 37°C for 90 minutes. After incubation, the content of the plate was discarded and washed twice with wash buffer, and then 100µl of biotin-labeled antibody was added into the wells, the plate was then covered and incubated for 60 minutes at 37°C. Later the content of the plate was discarded and washed thrice with wash buffer, then 100µl of HRP-streptavidin conjugate (SABC) working solution was pipetted into each well, which was then covered and incubated at 37°C for 30 minutes. After the incubation, the plate was washed with a wash buffer five times, allowing the wash buffer to stay for two minutes each time in the well. Next, 90µl of TMB substrate was added into each well, the plate was sealed and incubated at 37°C in the dark for fifteen minutes. Then, 50µl of acidic stop solution was added into each well. The color changed from blue to yellow immediately after the addition of the substrate. The absorbance was read in a microplate reader at 450nm immediately after adding the stop solution. Concentrations were extrapolated using a standard curve.

Serum phosphorylated-NF-κβ quantification 

Serum Human phosphorylated nuclear factor kappa B (NF-κB) concentrations were determined using ELISA Kit obtained from Wuhan Fine Biotech Co., Ltd, China (Batch No. H1950F033). All reagents and samples were allowed to equilibrate to room temperature (18-260C) and then shaken well. Each microtiter well was washed twice by dispensing 250µL of diluted wash buffer. Exactly 100µl of the prepared standard was aliquot into the standard wells while 100µl of sample dilution buffer was added into the blank well and 100µl of the sample were properly added into the test sample wells. The plate was sealed with a cover and incubated at 37°C for 90 minutes. After incubation the contents of the plate were discarded and washed twice with wash buffer, and then 100µl of biotin-labeled antibody was added into the wells, the plate was then covered and incubated for 60 minutes at 37°C. Later the contents of the plate were discarded and washed thrice with wash buffer, then 100µl of HRP-streptavidin conjugate (SABC) working solution was pipetted into each well, which was then covered and incubated at 37°C for 30 minutes. After incubation, the plate was washed with a wash buffer five times, allowing the wash buffer to stay in the well for two minutes each time. After that, 90µl of TMB substrate was added into each well, the plate was sealed and incubated at 37°C in the dark for fifteen minutes. Then 50µl of acidic stop solution was added into each well. The color changed from blue to yellow immediately after the addition of the substrate. The absorbance was read in a microplate reader at 450nm immediately after adding the stop solution. The concentrations were extrapolated using standard curve.

Determination of Serum Reduced Glutathione

Reduced glutathione (GSH) concentration was determined according to standard procedures [30,31]. The assay is based on the reaction of 5,5- dithiobisnitrobenzoic acid (DNTB) and reduced Glutathione (GSH). To 150µl of the sample, 1.5ml of 10% trichloroacetic acid (TCA) was added and then centrifuged at 1500 × g for 5 minutes. After which 1000µl of the supernatant was treated with 500µl of Ellman’s reagent and then 3ml of phosphate buffer (0.2M, pH 8.0) was added. The absorbance of the mixture was read at 412nm using a spectrophotometer. The concentration of GSH was obtained from the standard curve.

Determination Of Serum Malondialdehyde

Lipid peroxidation is evidenced by formation of TBARS which was measured using the modified method of 32 and described by 33. Lipid peroxidation generates peroxide intermediates which upon cleavage release malondialdehyde (MDA), a product which reacts with thiobarbituric acid (TBA), forming a MDA-TBA adduct that absorbs strongly at 535nm. Exactly 150µl of the samples was treated with TBA-TCA-HCL reagent (1:1:1 ratio) which was then placed in a water bath at 90°C for 60 minutes, later the mixture was cooled and centrifuged at 3000 rpm for 5 minutes and the absorbance of the pink supernatant (TBA-malondialdehyde complex) was then read at 535nm. Malondialdehyde formed was then calculated using the molar extinction coefficient of 1.56 × 10-5cm-1M-1

Data Analysis

Where appropriate, results were presented as the mean ± SD. Data was analyzed using a statistical software package (SPSS for windows, version 21, IBM Corporation, NY, USA). Statistical difference between means was analyzed using one-way ANOVA and Duncan post-hoc test was used to ascertain the degree of significance, while association studies and relationships were analyzed using Chi-square test and correlation analysis respectively. Results are significantly different at P ≤ 0.05.  

RESULTS

Socio-Demographic Characteristics Among Postmenopausal Breast Cancer Patients Attending Surgical Outpatient Clinic, ABUTH, Zaria

The socio-demographic characteristics of the histologically confirmed breast cancer patients showed that the majority (38.3%) of the breast cancer patients were within the age group of 45 – 50 years with mean age of 54.7±6.7 years while 26.67% were within the age group of 51 – 55 years. Among the breast cancer patients, 76.6% were married while 10% were widowed. Forty percent of respondents had tertiary education followed by secondary education with 30%. Majority of the breast cancer patients (55%) were of Hausa ethnic group and 60% were unemployed (Table 1). 

Classification of Breast Cancer Patients into Subtypes 

Breast cancer patients were classified into different subtypes based on the immunohistochemistry result (Table 2). Triple negative breast cancer (TNBC) was the most frequent accounting for 40% followed by Luminal A (31.7%), while HER2-enriched and Luminal B were 20% and 8.3% respectively. Hence 40% of the cases were ER-positive (Luminal A and Luminal B), whereas 60% are found to be ER-negative (HER2-enriched and triple negative). The result also showed that 83.3% had invasive carcinoma, no specific type NST-histological type and most of the patients presented at latter stages of the disease; stage III (53.3%) and stage IV (28.3%) (Table 2).

Association Between Breast Cancer and Possible Risk Factors Among Postmenopausal Breast Cancer Patients 

Association between possible risk factors and breast cancer subtypes showed significant association (χ2= 20.219, P= 0.017) between ethnicity and breast cancer subtypes (Table 3). However, the results showed no association (P>0.05) between breast cancer and marital status, age at menarche, age, alcohol consumption, birth control pills, pregnancy and body mass index (Table 3).

The association between possible risk factors and breast cancer severity indicated significant association between breast cancer severity and pregnancy (χ2= 25.671, P= 0.012) as well as breast cancer severity and body mass index (χ2= 25.278, P= 0.001), between breast cancer severity and marital status (χ2= 18.992, P= 0.025) (Table 4). In addition, there was no significant association (P>0.05) between breast cancer severity and age at menarche, age, alcohol consumption as well as the use of birth control pills (Table 4).

Serum Levels of Pro-inflammatory Markers (RANKL and NF-κB) among Postmenopausal Breast Cancer Patient 

The serum levels of RANKL and NF-κβ within the different molecular subtypes of breast cancer showed that the serum concentrations of RANKL were significantly (P<0.05) higher in Luminal A and Luminal B subtypes of when compared to apparently healthy controls (Figure 1a). Also, the concentration of RANKL was significantly (P<0.05) higher in HER2-enriched and triple negative subtypes of breast cancer when compared to the apparently healthy control group. Although, there was no significant (P>0.05) difference between luminal A subtypes and luminal B subtype as well as between HER2-enriched subtype and triple negative subtype of breast cancer. Generally, RANKL concentration is significantly (P<0.05) higher in breast cancer patients when compared to the apparently healthy individuals (Figure 1a).

NF-κβ concentration across the different subtypes of breast cancer, was significantly (P<0.05) higher in triple negative and HER2-enriched subtypes as compared to apparently healthy control (Figure 1a). The data also revealed that NF-κβ concentration was significantly (P<0.05) higher in luminal A and luminal B subtypes of breast cancer when compared with apparently healthy control. Similarly, NF-κβ concentration was significantly (P<0.05) higher in all breast cancer patients when compared to apparently healthy controls (Figure 1a). 

The serum concentration of RANKL and NF-κβ across different stages of breast cancer severity demonstrated a significant (P<0.05) increase in RANKL across stage I to stage IV (Figure 1b). However, there was no significant (P>0.05) difference between stage II and stage III as well as between stage III and stage IV. The result also revealed a significant (P<0.05) increase in NF-κβ concentration from stage I through stage IV. However, the increase was not significant (P>0.05) with respect to stages I and II as well as between stages II, III and IV (Figure 1b).

The serum RANKL and NF-κβ concentrations across different chemotherapy courses taken by the patients showed a slight decrease in RANKL concentration from 1st course through 6th course though the decrease was not significant (P>0.05)  (Figure 1c). The result also showed a significant (P<0.05) decrease in NF-κβ concentration in 3rd course when compared to 2nd course of chemotherapy, but there was no significant (P>0.05) difference with 1st course, 4th course, 5th course, and 6th course of the chemotherapy (Figure 1c).

Associations Between Serum RANKL & NF-Κβ Levels and Possible Risk Factors Among Postmenopausal Breast Cancer Patients

The association between possible risk factors and serum concentration of RANKL and NF-κβ were tested using Chi-square. The result indicated no association (P>0.05) between serum RANKL and the possible risk factors (Table 5). Moreover, no association between NF-κβ concentration and possible risk factors (age, pregnancy, birth control pills, alcohol consumption, and age at menarche, ethnicity, education level, occupation, and marital status).   

Serum Levels of some Oxidative Stress Biomarkers among postmenopausal Breast Cancer Patients 

The serum concentration of some oxidative stress markers (GHS and MDA) was presented in Figure 2a. The results showed no significant (P>0.05) difference among the four subtypes of breast cancer with respect to GSH concentration, but it is significantly (P<0.05) higher in breast cancer patients when compared to apparently healthy individuals. Furthermore, the serum MDA concentration was significantly (P<0.05) higher in Luminal A subtype when compared to HER2-enriched. Although higher Luminal A is not significant (P<0.05) when compared to triple negative as well as Luminal B subtypes of breast cancer. Serum malondialdehyde concentrations were significantly (P<0.05) higher among breast cancer patients when compared to apparently healthy individuals.

Serum oxidative stress markers across stages of breast cancer severity was presented in Figure 2b. The result showed an increase in MDA concentration from stage I to stage IV. However, stages III and IV have significantly (P<0.05) higher MDA level compared to stage I and II. Serum GSH level was decrease from stage I to IV, with Stages III and IV significantly (P<0.05) lower compared to stages I and II.

Figure 2c shows oxidative stress levels across different chemotherapy cycles. There was no significant (P>0.05) difference in MDA concentration across the different chemotherapy courses. GSH concentration decreases from 1st to 6th course. However, the reduction in GSH was significantly (P<0.05) lower in the 6th course compared to 1st, 2nd, 3rd, 4th, and 5th courses. It is also significantly (P<0.05) lower in the 4th and 5thcompared to 1st, 2nd, and 3rd courses.

Relationship Between Oxidative Stress Biomarkers and Inflammatory Markers Among Postmenopausal Breast Cancer Patients 

The relationship between some oxidative stress biomarkers (GSH & MDA) and the pro-inflammatory biomarkers (RANKL & NF-κB) of the breast cancer patients were tested using Pearson’s correlation (Table 6). The results showed that there were significant positive correlations between NF-κβ and RANKL (r = 0.916; P=0.001), NF-κβ and GSH (r = 0.695; P=0.001) and NF-κβ and MDA (r = 0.834; P=0.001). In addition, significant positive correlations were also observed between the level of RANKL and GSH (r = 0.842; P=0.001) and between RANKL and MDA (r = 0.939; P=0.001). There was also a significant positive relationship between serum GSH and MDA (r = 0.860; P=0.001).

DISCUSSION

Breast cancer incidence and mortality is characterized by variations in disease pathogenesis and treatment outcomes across racial and ethnic boundaries, pointing to a multifactorial basis for the disease 11. A number of factors could be responsible for the high mortality rate among breast cancer patients in West Africa and Nigeria in particular, necessitating research into socio-demographic patterns, breast cancer phenotypes and possible biochemical alterations in these patients 3. The modal age of the respondents from this study was 45 - 50 years, while the mean age was 54.7 ± 6.7 years and is similar to the age of breast cancer patients observed by a study carried out in South-West Nigeria 34. The immunohistochemistry data showed that triple negative breast cancer was the most common subtype among the patients and agrees with previous studies which showed that women of West African ancestry are more predisposed to the triple negative breast cancer phenotype 35. Majority of the patients were observed to be in stages III and IV, suggesting that the cancer was aggressive and possibly metastasized which agrees with the aggressive pattern of breast cancer pathogenesis observed in previous studies 34,36. 

Interestingly, the data from our study showed no association between breast cancer and marital status, age at menarche, and body mass index which are established risk factors for breast cancer predisposition 37. This might be due to the fact that some of these risk factors are modifiable. We also found no association between history of alcohol consumption, use of birth control pills and breast cancer risk which disagrees with a previously reported study carried out among breast cancer patients in West Africa 38. This could be due to the lifestyle of the women in the area as most of them are from local communities and are mostly not exposed to alcohol use as well as birth control pills for personal and religious reasons. 

Moreover, the serum level of Receptor Activator of Nuclear factor Kappa beta Ligand (RANKL) among the breast cancer patients was significantly higher when compared to apparently healthy control subjects, agreeing with previous studies which showed that RANKL plays significant roles in mammary epithelial cell proliferation and is dysregulated during breast cancer 39. The data also showed that among the patients, RANKL was higher in patients with triple negative phenotype compared to apparently control subject which is reportedly associated with worse clinical outcomes and bone metastasis in breast cancer patients with higher RANKL activity 40. Also, serum NF-kB levels were significantly higher in breast cancer patients when compared to apparently healthy control, agreeing with the observation that NF-kB activity correlates with increased disease severity among breast cancer patients  41. The serum levels of both RANKL and NF-kB were found to correlate with disease severity and were higher in patients at later stages of the disease, further suggesting that these proteins could have prognostic potential.

Oxidative stress is implicated in breast cancer pathogenesis 42, and our data showed significantly higher level of reduced glutathione (GSH) and higher level of malondialdehyde (MDA) in breast cancer patients when compared to apparently healthy control subjects. MDA was more elevated while GSH was more depleted in stage III and IV patients suggesting that oxidative stress correlated with disease progression which is similar to previously reported studies 43.

CONCLUSION

Breast cancer patients studied in ABUTH, North-Western Nigeria showed no association between breast cancer and birth control pills and alcohol. However, serum levels of RANKL and NF-kβ were elevated in the patients and were higher as the disease progressed pointing to their strong procarcinogenic activity due to their role in tumor cells proliferation, survival, angiogenesis, inflammation, and metastasis. Malondialdehyde was elevated while glutathione was depleted in the patients and was more evident as the disease progressed, suggesting oxidative stress and a possible need for dietary awareness among the patients. Further cohort studies are needed to examine if similar patterns of RANKL and NF-kβ levels will be observed in patients viz a viz disease progression or response to therapy to determine the possible prognostic potential of RANKL and NF-kβ in postmenopausal breast cancer patients.

Abbreviations:

RANKL= receptor activator of nuclear factor kappa-beta ligand, NF-κβ = nuclear factor kappa-beta, GSH = reduced glutathione, MDA = malondialdehyde, ABUTH = Ahmadu Bello University Teaching Hospital 

Acknowledgement:

we would like to thank the subjects for consenting to be part of the work, and Ahmadu Bello University Zaria for providing the conducive environment and necessary equipment to carry out the research. 

Conflict of Interest:

 We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Data Availability Statement

Data will be available and share on request.

 

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