Clinical Data
A total of 56 breast cancer patients were studied over four years, from 2018 to 2021. Out of 56 patients, 31(55.35%) were under 50 and 25 (44.65%) were over 50 years old. The average age of patients was 51.6 ± 12.62 (Range 32-80yrs). Most patients had an active lifestyle, accounting for 42 (75%) of the total number of patients, whereas 14 (25%) had a sedentary lifestyle. Out of all patients, 06 (10.7%) had a family history of either breast cancer or other solid malignancy and 50 (89.3%) patients had no family history of any malignancy. Most of the patients had normal body mass 42 (75%), except a few patients who were lean 05 (8.93%) and obese 09 (16.07%). Only 21 (37.5%) of the patients were from Kashmir's urban districts, while 35 (62.5%) were from rural areas (Table 1).
Table 1
The various characteristic features of Breast cancer patients
Patient characteristics
|
Controls
|
Cases
|
p-Value
|
Age
≤ 50
> 50
|
41 (73.21%)
15 (26.79%)
|
31 (55.35%)
25 (44.65%)
|
0.0486
|
Gender
Male
Female
|
7 (12.5%)
49 (87.5%)
|
02 (3.57%)
54 (96.43%)
|
0.8221
|
Dwelling
Rural
Urban
|
40 (71.42%)
16 (28.58%)
|
35(62.5%)
21(37.5%)
|
0.3151
|
Life Style
Active
Sedentary
|
47 (83.92%)
09 (16.07%)
|
42 (75%)
14 (25%)
|
0.2421
|
Lymph Node Status
Positive
Negative
|
|
33 (58.92%)
23 (41.07%)
|
|
Family History
With History
Without History
|
-
-
|
06 (10.71%)
50 (89.29%)
|
-
-
|
Necrosis
Present
Absent
|
-
-
|
26 (46.42%)
30 (53.57%)
|
-
-
|
Menopausal Status
Premenopausal
Postmenopausal
|
28 (57.1%)
21 (42.85%)
|
24 (44.44%)
30 (55.55%)
|
0.1979
|
HIF-1 α, VEGFA and HK-I expression in BC
HIF-1α (p=0.0010) and VEGFA (p=0.0119) transcriptional expression was found to behigher in tumor tissues and blood of the patients than in surrounding normal/peritumor tissues and normal blood samples, respectively, whereas HK-I (p=0.9842) was also higher in tumor tissues but not statistically significant. HIF-1α (p=0.0111) and VEGFA (p=0.0078) relative mRNA expression were greater in TNBC and Her2+ breast tumors (Figure 1).
The HIF-1α expression in the BC subgroups was significantly higher (p = 0.0176), with the TNBC group having the highest expression and the ER+/PR + group having expression value between TNBC and HER2+. The expression of VEGFA was also found to be higher in TNBC (p = 0.0119) and HER2+ (p = 0.0078) sub-group. While as the expression of HK-I was significantly higher in ER/PR +, HER2– group (p = 0.0106) than other sub groups of BC respectively (Fig. 1).
HIF-1α, VEGFA and HK-I immunoexpression in breast carcinoma cases
Positive HIF-1α immuno-expression was observed in 33 (62.26%) of the 53 samples, whereas positive VEGFA expression was found in 38 (71.69%) of the samples, and this was perceived to be statistically linked with higher histopathological grade (p = 0.0006) and positive lymph node metastasis (p = 0.031).The HK-1 immuno-expression was positive in 27 (50.9%) of the 53 samples. HIF-1α immuno-expression was predominantly found in the nucleus. However, it was also seen in cytoplasm, where it may operate as a transcription factor, VEGF-A and HK-I were mostly found in the cytoplasm (Figs. 2, 3 and 4).
Association Between Hif-1α, Vegf-a And Hk-i Expression And Clinicopathological Variables
The increased expression of HIF-1α is known to increase the expression of VEGFA and HK-I. Results of the Spearman correlation indicated that there is a non-significant yet positive relationship between HIF-1α and VEGFA, (r =0.208, p =0.186) HIF-1α and HK-I (r =0.121, p =0.447) and a non-significant, negative relationship between HK-I and VEGFA (r=0.039, p =0.806) (Figure 5).
Thus, without reaching any conclusive statistical significance, the mRNA expression of HIF-1α was evaluated, and it was found to be higher in the lower age group (≤ 50 years), moreover the mRNA expression of VEGFA and HK-I was found to be statistically significant in the higher age group (≥ 50 years) (Fig. 6A). The expression of HIF-1α was also statistically higher in pre-menopausal women, whereas VEGFA expression was higher in post-menopausal women and HK-I was slightly higher in pre-menopausal group without reaching any statistical significance (Fig. 6B). The transcriptional expression levels of HIF-1α, VEGFA and HK-I werefound to be higher with increasing grade (Grade II & III) (Fig. 6C).
Correlations between HIF-1α, VEGF-A, HK-I, and clinicopathological data were examined using χ2 test to calculate the clinical prominence in BC. Relatively high continuous mRNA expression levels of VEGF-A andHIF-1α were not linked to any of theclinicopathological parameters, while HK-I expression was significantly related to different subtypes of BC (p = 0.036) and necrosis of tumor tissue (p = 0.029) & in Table 2.
Table 2
Correlation coefficients between HIF-1α, VEGF-A and HK-I expression and clinicopathological parameters in breast carcinoma.
|
Patient characteristics
|
HIF-1α
|
p-value
|
VEGFA
|
p-value
|
HK1
|
p-value
|
Low
|
High
|
Low
|
High
|
Low
|
High
|
Age
|
≤ 50
> 50
|
11
|
20
|
0.343
|
8
|
23
|
0.608
|
13
|
18
|
0.295
|
12
|
13
|
5
|
20
|
14
|
11
|
|
Histological Subtype
|
Ductal
Lobular
Others
|
16
|
24
|
0.276
|
13
|
27
|
0.559
|
17
|
23
|
0.954
|
2
|
2
|
1
|
3
|
2
|
2
|
2
|
10
|
2
|
10
|
5
|
7
|
Family History
|
Yes
No
|
1
|
5
|
0.344
|
2
|
4
|
0.534
|
3
|
3
|
0.852
|
18
|
32
|
11
|
39
|
23
|
27
|
Lymph Node
|
Positive
Negative
|
10
|
23
|
0.311
|
9
|
24
|
0.921
|
15
|
18
|
0.415
|
10
|
13
|
6
|
17
|
13
|
10
|
Grade
|
I
II
III
|
2
|
5
|
0.834
|
2
|
5
|
0.159
|
5
|
2
|
0.296
|
11
|
17
|
11
|
17
|
12
|
16
|
7
|
14
|
3
|
18
|
8
|
13
|
Subtypes
ER, PR,Her2
|
+ + +
+ + -
- - +
- - -
|
6
|
2
|
0.097
|
3
|
5
|
0.259
|
5
|
3
|
0.036
|
12
|
13
|
11
|
14
|
16
|
9
|
3
|
9
|
2
|
10
|
2
|
10
|
3
|
8
|
2
|
9
|
4
|
7
|
Menopausal
|
No
Yes
|
10
|
14
|
0.528
|
8
|
16
|
0.266
|
10
|
14
|
0.393
|
10
|
20
|
6
|
24
|
16
|
14
|
Necrosis
|
Absent
Present
|
12
|
18
|
0.861
|
7
|
23
|
0.351
|
18
|
12
|
0.029
|
11
|
15
|
9
|
17
|
8
|
18
|
Stage
|
I
II
III
|
3
|
4
|
0.631
|
2
|
5
|
0.933
|
5
|
2
|
0.406
|
9
|
21
|
8
|
22
|
13
|
17
|
8
|
11
|
6
|
13
|
9
|
10
|
Prognostic value of HIF-1α, VEGFA and HK-I
The survival analysis was performed by using Kaplan–Meier approach to assess the relationship between overall survival rates and mRNA expression of HIF-1α, VEGFA and HK-I. The overall survival rates for high and low expression of HIF-1α, VEGFA and HK-I were 71.1% & 64%, 65.71% & 84%, and 70.74% & 62.5% respectively. The cox-regression hazard model was used to identify independent prognostic determinants. High levels of VEGF-A were also linked to poor prognosis for BC (HR=1.88). While HIF-1α (HR=0.808) and HK-I (0.7884) levels were not linked with the prognosis. The study found that high VEGFA expression, lymph node involvement, TNBC type, advanced grade, and stage (II & III) are independent prognostic variables for BC (Figure 7).
During the survival analysis, it was observed that patients with HIF-1𝛼 and HK-I overexpression had favorable prognosis and patients with VEGF-A overexpression had a worse prognosis. In oncological research, a lot of work is being put into targeting metabolic pathways that are active in cancerous cells. Hypoxia is a well-recognised process to accelerate glycolysis and promotes the expression of various metabolism-related genes;therefore specifically targeting biochemical pathways might kill hypoxic cells or make them more sensitive to conventional chemotherapy or a specialized targeted treatment. This may pave theway towards personalized medicine.
Heat map plot analysis also represented the differential expression of the HIF-1α, VEGF-A, and HK-I along with the classification based on the ER, PR and Her2. Columns of the plot represent the genes and the rows represent samples (Fig. 8A-E). Moreover, the gene-gene interaction analysis of HIF-1α, VEGF-A, and HK-I revealed that EPAS,CUL2,SLC2A1, FLT1, ARNT, HIF1AN and VHL showed maximum interaction (Fig. 9). These genes might play a role in BC progression and could be explored for future studies.