A novel role of kynureninase in the growth control of breast cancer cells and its relationships with breast cancer

Abstract Breast cancer is the most common malignancy among women worldwide. Kynureninase (KYNU) located in 2q22.2, which was associated with tryptophan utilization and metabolic diseases including cardiac, renal and limb defects syndrome 2. However, the role of KYNU in breast cancer (BC) development remains unclear. The expression of KYNU was examined by immunohistochemistry (IHC) in 137 primary BC tissues, and the correlation of KYNU expression with clinical pathological characteristics and the biomarkers (ER, PR, HER2, E‐cad and Ki‐67) was analysed. The role of KYNU in cancer cell proliferation, tumour growth and development was evaluated by MTT assay, soft agar colony formation assay and xenograft mouse models. Among 137 primary BC tissues, 46.7% (64/137) had high KYNU expression (IHC scores >4) while 53.3% (73/137) had low KYNU expression (IHC scores ≤4). The expression of KYNU was positively correlated with the expressions of ER (P = .002), PR (P = .007) and E‐cad (P = .03), while negatively associated with tumour grade (P = .008), tumour stage (P < .001) and the expressions of HER2 (P = .04) and Ki‐67 (P = .019). Overexpression of KYNU significantly inhibited cell proliferation in cell culture, colony formation in soft agar and xenograft BC development in NOD/SCID mice. Kynureninase suppresses BC cell proliferation, tumour growth and development. Kynureninase may function as a tumour suppressor in BC.


| INTRODUC TI ON
Breast cancer (BC) is the most common diagnosed cancer and the fifth leading cause of cancer death among women in China. 1 Although the rapid development of target therapy and immunotherapy in recent years, surgery, hormone therapy, radiotherapy and chemotherapy are still the effective options for the treatment of BC. 2 The 5-year survival rate of early-stage breast cancer (EBC) patients in China is about 58%-78%. 3 Breast cancer is a clinically heterogeneous disease. However, BC cells from the cancerous lump may have spread to other organs of patients after different kinds of treatment (a mastectomy, radiotherapy and chemotherapy). Although tumour size, histopathological grades and clinical stages are important indicators for clinical management of BC, it is a common phenomenon that histologically similar tumours may have different prognoses and respond to therapy differently, due to the molecular difference among the histologically similar tumours. Kynureninase (KYNU) or L-kynurenine hydrolase belongs to the kynureninase family. Kynureninase gene is located at 2q22. 2. 4 KYNU is a pyridoxal-5′-phosphate (pyridoxal-P)-dependent enzyme that catalyses the cleavage of L-kynurenine and L-3-hydroxykynurenine into anthranilic and 3-hydroxyanthranilic acids, respectively. Kynureninase is involved in the biosynthesis of NAD cofactors from tryptophan through the kynurenine pathway. 5 Kynureninase was associated with tryptophan utilization and metabolic diseases. It has been reported that KYNU is associated with metabolic neurological, 6 cardiac 7 and renal disease. 8 Inhibition of the kynurenine pathway protects against reactive microglial-associated reductions in the complexity of primary cortical neurons. 9 KYNU has been reported to be down-regulated in the highly aggressive osteosarcoma cell lines. 10 The association of KYNU with cancer is rarely reported, and the role of KYNU in cancer development is unclear.
Here, we report that the expression of KYNU is negatively correlated with clinical BC histological grades and tumour stages.
Overexpression of KYNU inhibits BC cell proliferation, colony formation and xenograft tumour development in mouse models.
Kynureninase may function as a tumour suppressor in BC.

| Immunohistochemistry (IHC) analysis
For IHC, briefly, 4-μm-thick paraffin-embedded tissue sections were treated with an antigen retrieval solution (eBioscience, #00-4955-58) and stained by using a Vectastain ® abc kit, according to the manufacturer's protocol (Vector Laboratories, # PK-4000). The tissue sections were incubated with the primary antibodies ( for Ki67, if ≥15% of the nuclei were stained, sample was classified as Ki67-positive (high) expression. 13 E-cad expression was considered positive if positive cells is equal to or more than 50% continuous membrane staining was present in the breast cancer cells, and negative or low expression if less than 50%, which was the median percentage observed in the included subjects. 13 The KYNU protein was found to be expressed primarily in the cytoplasm of tumour cells. The cytoplasm staining fraction (CF) was assigned a score of 0 (0%-5%), 1 (5%-25%), 2 (26%-50%), 3 (51%-75%) or 4 (>75%), and cytoplasm staining intensity (CI) was noted as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). Subsequently, a combined Cytoplasm TA B L E 1 Antibodies and staining conditions in the study For statistical analyses, the cut-off values for KYNU expression were chosen on the basis of heterogeneity using the log-rank test for OS.
The optimal cut-off value was determined as low (scores ≤4) or high (scores >4) KYNU expression.

| Western blot analysis
Cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. Immunostaining was done using antibodies specific for KYNU (GeneTex, Lot No. 33291). Chemiluminescence detection was performed by using the Pierce ECL Western blotting substrate (Thermo Scientific).

| The differential expression of KYNU in breast cancer and benign cells
Among all sections analysed, we found that KYNU protein was highly expressed both in mammary ductal carcinoma cells ( Figure 1A,C) and in benign myoepithelium cells ( Figure 1D), but not in lymphocyte and benign mammary gland luminar cells ( Figure 1C,B,D).
The expression of KYNU was very high in well-differentiated BC cells ( Figure 1E) and very low in poor-different BC cells ( Figure 1F).

| The expression of KYNU is positively correlated with BC differentiation but negatively with BC tumour grade and tumour stage
As shown in Figure Figure 2D), high in T1 BC ( Figure 2E), while its expression was low in T2 BC ( Figure 2F) and very low in T3 BC ( Figure 2G). The expression of KYNU was reversely related to tumour stage (T stage) ( Figure 2I).

| The association of KYNU expression with breast cancer clinicopathological characteristics
Typical clinicopathological parameters and immunohistochemical staining of low or high KYNU expression are shown in Table 2.
However, the expression of KYNU was not significantly related to patients' age (year) or N-stage(N) of BC.

| The association of KYNU expression with other biomarkers of BC
We further analysed the correlation of KYNU expression with expression of other biomarkers. We found that the expression of KYNU was positively associated with the expression of ER, PR and E-cadherin (Table 2, Figure 3A-C), respectively, while negatively related to the expression of HER2 (Table 2, Figure 4D) and Ki-67 (Table 2, Figure 3E).

| Overexpression of KYNU inhibits BC proliferation in culture and colony formation in soft agar
To

| KYNU suppresses BC xenograft tumour development in mouse models
To further investigate the role of KYNU in BC development, 5 ×  Figure 5A). We found that tumour weight was significantly higher in control mice than in KYNU-OE mice ( Figure 5B).
MDA-MB-231 cells with overexpression of KYNU had significant slower tumour development in mice than control cells ( Figure 5A,B), suggesting overexpression of KYNU inhibits BC development.

| D ISCUSS I ON
Breast cancer (BC) is the most common cancer in females and the leading cause of death worldwide, 14 which are clinically heterogeneous. Because of heterogeneous, clinically BC may be respond poorly to neoadjuvant chemotherapy, or it is respond well to endocrine therapy. 15 After a loss or reduction of the suppressor genes in its function, normal cells may become into cancer cells with the combination of other genetic changes. 16 Scientists have learned more about the molecular changes that lead to cancer. The loss of these suppressor genes may be even more important than protooncogene/oncogene activation for the formation of many kinds of human cancer cells. 17 19 Time-lapse microscopy revealed that myoepithelial cells collectively restrain and reinternalize invading Twist1+luminal cells. 19 Barrier function correlated with myoepithelial abundance and required the expression of αsmooth muscle actin and P-cadherin. 19 The data showed the concept of the myoepithelium as a dynamic barrier to luminal dissemination  Here, we report that the expression of KYNU is negatively correlated with clinical BC histological grades and tumour stages.
Overexpression of KYNU inhibits BC cell proliferation, colony formation and xenograft tumour development in mouse models.
What do we know, it is not reported that KYNU is related to human breast cancer. Our data showed that KYNU may function as a tumour suppressor in BC. Therefore, KYNU is a promising therapeutic target of BC, but it need take a more further study of KYNU. Here, we only had a try for a Sea horse test ( Figure S1). In the future, we will study more about the KYNU molecular changes that lead to breast cancer.

CO N FLI C T O F I NTE R E S T S
All authors declare that they have no competing interests. Tumour weight was significantly higher in control mice than in KYNU-OE mice (B). (*P < .05, **P < .001)