Expression of RPL32 predicts response to anthracycline-based neoadjuvant chemotherapy in triple-negative breast cancer

doi:10.21203/rs.3.rs-1539834/v1

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Research Article

Expression of RPL32 predicts response to anthracycline-based neoadjuvant chemotherapy in triple-negative breast cancer

https://doi.org/10.21203/rs.3.rs-1539834/v1

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Background

Ribosomal protein 32 (RPL32) plays an important role in carcinogenesis. Previous studies have shown that RPL32 is closely related to the occurrence and development of tumor tissues. Nevertheless, the clinicopathological features and functional role of RPL32 in triple-negative breast cancer(TNBC)patients treated with anthracycline-based neoadjuvant chemotherapy (NAC) chemotherapy are still unclear.

Methods

In order to fill this gap, we systematically analyzed the expression of RPL32 in cancer tissues of 96 TNBC patients treated with anthracycline-based NAC and the relationship between RPL32 and clinical features of TNBC. We also discussed the relationship between RPL32 expression and adriamycin resistance in TNBC cells. KM analysis was used to determine the relationship between RPL32 and DFS, DDFS and OS. Univariate and multivariate logistics analysis was used to evaluate whether RPL32 was an independent factor of pCR in TNBC patients.

Results

The IHC staining showed 30 (31.25%) RPL32 protein was located in the TNBC cytoplasm. The RPL32 expression was linked with MP grading, lymph node metastasis, and whether achieved pCR. In vitro, RPL32 up-regulation substantially augmented the ability of TNBC cells to form lines colonies. RPL32 overexpression in TNBC cells enhanced Akt phosphorylation. RPL32 overexpression was shown to be correlated to lower DFS, DDFS, and OS. Univariate and multivariate analyses showed that expression of RPL32 was the independent factor associated with pCR.

Conclusion

The findings suggest that RPL32 may be a potential marker of TNBC patients' response to NAC and has clinical significance as a target for improving chemosensitivity.

RPL32

Triple-negative breast cancer

Neoadjuvant chemotherapy

anthracycline

Triple-negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype having a poor prognosis. Chemotherapy is an effective adjuvant in the treatment of TNBC. Neoadjuvant chemotherapy (NAC), is commonly used to shrink tumors before surgery[1]. That can increase the chances of breast-conserving surgery. It can also provide an in vivo analysis of tumor response to the treatment.

A large patients population with TNBC needs NAC[2]. TNBC is managed with anthracycline and taxane-based chemotherapy, which is considered the gold standard[3]. At least one-third of the patients with stages II and III TNBC can achieve a pathological complete response (pCR) after anthracycline and taxane-based NAC[4, 5]. However, drug resistance cannot be ignored, which can lead to tumor progression and unnecessary exposure to drug side effects. More severely, patients may miss out on the chance to get effective treatment therapy. Therefore, the identification of biological markers, that can accuratly predict of response in TNBC, is needed.

Ribosomal proteins (RP) are ribosomal components involved in protein translation and ribosome assembly[6]. They play an important role mainly in catalyzing protein translation and carcinogenesis. Some studies have shown the tissue-specific expression of certain RPs that can affect the composition of ribosomes, the processing of ribosomal RNA, and the regulation of translation[7]. Previously, it has been found that many kinds of human cancers, including the bladder, brain, pancreatic, and other tissue cancers, have multiple individual RPL abnormalities[8–14]. RPL32, a component of the 60S subunit, encodes an RP. Previous research has shown that RPL32 is linked to cancer growth and metastasis[15–18]. A recent study found that the silencing of RPL32 could pointedly decrease the metastasis of breast cancer, therefore indicating that RPL32 might be a potential diagnostic marker for BC[19]. Though, the relationship between RPL32 and clinical feature in TNBC and the function of RPL32 in TNBC drug resistance is still unclear.

In the research, RPL32 expression was evaluated in TNBC tissues and the cell line. We aimed to study the role of RPL32 as a predictive marker of TNBC patients undergoing anthracycline-based NAC. The function of RPL32 in drug resistance of TNBC was also studied.

2.1 Patient Samples

We examined a cohort of 96 patients with TNBC who had anthracycline-based neoadjuvant chemotherapy followed by surgical resection, with core biopsies taken before the first cycle of chemotherapy. Benign breast tissue samples were obtained from 22 women who underwent cosmetic surgery. We collected samples between January 2014 and January 2021. Prior to starting chemotherapy, all patients had a bilateral mammogram, a bilateral breast ultrasound, and drainage lymph node examination. Blood biochemistry, bone scan, chest CT, and abdominal ultrasound were performed before NAC to assess organ function and rule out metastatic disease. Patients with metastatic disease, inflammatory breast cancer, or male breast cancer were excluded from this study. Magnetic Resonance Imaging (MRI) was used for the findings of breast tumor size and metastases of the axillary lymph node. AJCC staging system (seventh version) was used for staging of patients were. Patients with BC were given doxorubicin (60 mg/m²) and cyclophosphamide (600 mg/m²) as neoadjuvant treatment. Every three weeks, repeated the cycle four times. Docetaxel (80–100 mg/m²) was used as a follow-up. Repeated 4 cycles every 3 weeks. The medical ethics committee of Harbin Medical University's Cancer Hospital gave its approval to the data collection. After completing 8 rounds of neoadjuvant chemotherapy, resection was conducted within 1 to 2 weeks.

2.2Assessment of the response to neoadjuvant chemotherapy

During neoadjuvant chemotherapy, a re-examination was performed every 2 cycles of chemotherapy to assess the effect of treatment. According to the therapy outcome, the patients were grouped according to RECIST (Solid Tumor Response Evaluation Criteria, RECIST 1.1) as complete response (CR), partial response (PR), disease stability (SD), and disease progression (PD). Pathological complete response (pCR) is generally used to evaluate the effect of neoadjuvant therapy. The pathology of pCR is defined as carcinoma in situ or breast lesions and regional lymph nodes free of residual invasive cancer cells post-chemotherapy . The proportion of tumor cells reduced after treatment and was divided into five grades by Miller-Payne (MP) grading, with grade 5 indicating no tumor-infiltrating cells.

2.3 Immunohistochemical staining.

This research includes 96 paraffin-embedded core needle biopsies of invasive breast cancer before neoadjuvant chemotherapy. The immunohistochemistry was performed according to the instructions. The tissues were treated with Anti-RPL32 primary antibody (1:1,000, dilution) (Abcam, cat. no. ab229758) in 1% BSA/PBS solution and overnight incubation at 4°C. The following day, the tissues were treated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:500) (Bioworld Technology, Inc., cat. nos. BS13278) for 1-hour at room temperature. After that, the color was developed using an immunoPure Metal enhanced Diaminobenzidine (DAB) Substrate kit (Thermo Scientific). The expression of RPL32 was determined by grading the cytoplasmic staining of brownish or yellowish granules as follows: I) 0, background staining; II) faint staining; III) moderate staining, and IV) intense staining. Positive staining regions incomplete tissue sections were also rated as follows: I) 0, < 5%; II) 1, 5‑25%; III) 2, 26‑50%; IV) 3, 51‑75%; and V) 4, 76‑100%. Negative and positive stainings were determined as 0-2 and ≥ 3, respectively, when combined with these two parameters^[19]^.The stained tissue samples were scored by two independent blinded investigators with a semi-quantitative scale.

2.4 Cell culture

The MDA-MB-231/ADR and BT-549/ADR cells lines were acquired from Cell Bank, Chinese Academy of Sciences (Shanghai, China). The cultures were produced by stimulating the parental cell lines with increasing doses of adriamycin. MDA-MB-231/ADR and BT-549/ADR cell lines were cultured for around 2 weeks in adriamycin-free RPMI medium after being incubated in 1.0 g/mL ADM-containing RPMI 1640 medium. The cells were cultured in humidified conditions at 5% CO₂ and 37°C and harvested in the logarithmic phase of growth.

2.5 CCK8 Assay

After inoculating cells on 96-well plates, at 5 × 10³ cells per well, for two days, 10 μL of CCK-8 reagent (DOJINDO, Japan) was then added. The cell was then incubated at 37 °C for 2-hours and the absorbance at 450 nm was measured through a microplate reader (Multiskan Mk3, Thermo Lab Systems, Finland).

2.6 Colony Formation and Apoptosis assay

HCC cells at density 1 x 10³ were inoculated in six-well plates and grown for 2 weeks. The cell colonies were then fixed by 4% formaldehyde and were stained with 1% crystal violet (Sigma). In each plate, the visible cell colonies were counted.

The cells were cultured for 24 hours in 6-well plates, before being transfected with the corresponding plasmids, to measure the apoptosis. The cells were collected after 24-36 hours, washed three times with ice-cold PBS, and a 500 μL binding buffer was used to resuspend the cells. Annexin V/FITC and Propidium iodide (PI) was used to stain cells in the dark condition for 10 min, and CytoFLEX was used to analyze the stained cells (BECKMAN COULTER).

2.7 Immunoblotting

RIPA lysis buffer was added to the cells for extraction of total proteins of the cell, which were then run on an 8% SDS-PAGE, and transferred the gel to PVDF membranes (Millipore). After blocking, the PVDF membrane was incubated with the primary antibody, overnight. The following day, the cells were then incubated with an HRP-conjugated secondary antibody. The target bands were quantified through the ECL kit (Bioworld).

2.8 Transfection

shRNA for RPL32, pcDNA3.1-RPL32, the scrambled oligonucleotides (shNC), and empty pcDNA3.1 vector (Vector) were acquired from Shanghai Biosmedi Co., Ltd. (Shanghai, China). Cells were grown up to 70–80 % confluency before being transfected, and transfected thorough Invitrogen's Lipofectamine 3000 Transfection Reagent. Subsequently, the cells were harvested after 48 hours of transfection for use in the next experiments.

2.9 Statistical Analysis

The SPSS software (SPSS ver23.0, USA) was used for all statistical analyses. The data were analyzed using a student’s t-test or one-way analysis of variance (ANOVA). The chi-square test was used to compare the count data. All the data ware represented as mean ± standard deviation (SD), Kaplan-Meier analysis was used to calculate the relationship between OS (Overall Survival), DDFS (distant disease-free Survival), DFS (disease-free survival), and RPL32 expression. The differences between groups were evaluated by log-rank. Univariate and multivariate logistics analysis was employed to assess whether RPL32 expression levels were an independent factor in TNBC patients. The statistically significance was considered as p-value (two-sided) < .05.

3.1 RPL32 expression in pre-neoadjuvant core needle biopsies of aggressive breast cancer

IHC was used to study the expression of RPL32 in TNBC tissues. The RPL32 protein in these tissues was located in the cytoplasm. There was a variable expression among the 96 breast cancer tissues, 30 (31.25%) were being positive(Fig. 1A), 66 (68.75%) were negative(Fig. 1B) for RPL32 protein. Whereas, no expression was observed in normal 22 samples of breast tissue(Fig. 1C).

3.2 Relationship between RPL32 expression and clinical features in TNBC patients

The RPL32 expression in TNBC tissues showed no assoscation with patient age, tumor grade, tumor size, clinical staging, ECOG, and Ki67 expression (p = .124, p = .612, p = .447, p =.156, p = .137, p = .372 , respectively) . Whereas, the expression of RPL32 was found to be correlated with MP grading, lymph node metastasis, and whether achieved pCR (p = .029, p = .027, p = .033) (Table 1).

3.3 RPL32 upregulation promotes adriamycin chemoresistance in TNBC cells

To test the hypothesis, TNBC cell lines MDA-MB-231 and TB549 that stably express RPL32 were constructed. RPL32 levels were considerably higher in MDA-MB-231 and TB549 cell lines, according to western blot analysis as shown in Figure 2A. Upregulation of RPL32 significantly improved the capacity of TNBC cell lines MDA-MB-231 and TB549 to form colonies (Figure 2B). Furthermore, in RPL32 overexpressing cells, the half-maximum inhibitory concentration (IC50) of adriamycin was considerably increased (Figure 2 C-D). In RPL32 overexpressing cells treated with Adriamycin, FACS analysis revealed reduced apoptotic rates (Figure 2E). According to our findings, the RPL32 has a critical role in adriamycin chemoresistance in TNBC.

3.4 Silencing of RPL32 induces adriamycin chemosensitivity in TNBC cells.

RPL32-silenced stable cell lines (MDA-MB-231 and TB549) were developed to further investigate the role of RPL32 in the development of TNBC cell chemoresistance (Figure 3A). Downregulation of RPL32 remarkably decreased the colony formation ability of MDA-MB-231 and TB549 cell lines (Figure 3B). In TNBC cell lines, downregulation of RPL32 reduced the IC50 value of adriamycin (Figure 3C-D). Furthermore, following adriamycin treatment, the apoptosis in RPL32-knockdown cells was greater than in vector control cells (Figure 3E). Finally, RPL32 knockdown improves TNBC cancer cells' chemosensitivity to adriamycin therapy.

3.5 RPL32 activates the Akt signaling pathway in TNBC cells.

The western blot analysis showed that RPL32 knockdown increased the apoptosis, as shown by higher expression of cleaved caspase-3 and Bax proteins, and a decrease in Bcl2 expression, while RPL32 overexpression induced the opposite effects (Figure 4A). In different cancer types, constitutive activation of the Akt signaling pathway provides chemoresistance[20, 21]. Immunoblotting analysis was used to assess the influence of RPL32 on the activation of the Akt signaling pathway to determine if RPL32 enhances chemoresistance through regulation of the Akt pathway. RPL32 overexpression enhanced (but RPL32 knockdown suppressed) Akt phosphorylation, as revealed by our findings (Figure 4B). RPL32-upregulated cells were treated with a combination of adriamycin and vehicle, or Adriamycin with the Akt inhibitor MK-2206, to see whether RPL32-induced chemoresistance was reliant on the Akt pathway. Our findings showed that MK-2206 abolished RPL32 overexpression-repressed apoptotic level in TNBC cells (Figure 4C), signifying that Akt inhibitor renders the sensitivity of TNBC cells to adriamycin therapy.

3.6 High expression of RPL32 is related to poor prognosis of neoadjuvant chemotherapy in TNBC

The survival status of 96 patients with TNBC who had neoadjuvant treatment was analyzed using the Kaplan-Meier method. The findings showed that Age, tumor grade and size, clinical stage, ECOG score, and Ki-67 index had little effect on the prognosis of patients, while lymph node metastasis, MP grade, and patient's response to chemotherapy were correlated with their prognosis. High RPL32 expression was related to a lower DFS (median DFS, 44 months vs 33 months, p = .041), as well as a lower DDFS (median DDFS, 47 months vs 36 months, p = .044). Similarly, High expression of RPL32 was significantly correlated with lower OS (median OS, 52 months vs 40 months, p = .041). These findings suggest that increased RPL32 expression is correlated to a poor prognosis in TNBC patients receiving neoadjuvant chemotherapy.

3.7 Univariate and multivariate analyses of the relationships between RPL32 expression,and clinicopathological findings

In univariate analyses based upon pCR, we found a significant association between Ki-67, p53, and RPL32 expression and pCR. In multiple regression analysis, p53 (odds ratio, 0.313; p = .032) expression and RPL32 (odds ratio, 0.254; p = .026) expression were the independent factors associated with pCR (Table 2).

NAC is recommended in a large proportion of locally advanced patients due to the aggressive nature of TNBC and the poor prognosis of most patients. Anthracycline-taxane regimens are considered standard treatment for TNBC. It should be noted that only a small number of patients with TNBC can achieve pCR in neoadjuvant therapy, but the survival of patients who achieve pCR is effectively prolonged[22, 23]. There is an unmet need to find out accurate biomarkers to predict TNBC response to anthracycline chemotherapy in NAC to reduce the use of toxic drugs, to assist clinicians in tailoring treatment methods to individual patients, and to enable more patients to achieve pCR in NAC.

RPL32 has been linked to carcinogenesis and the progression of various cancers. RPL32 was shown to have a good prediction accuracy when it came to SF3B1, one of the most commonly mutated genes in chronic B-lymphocytic leukemia (CLL)[24]. RPL32 is increased in late-passage androgen-independent (LNCaP-C81) cells compared to early-passage androgen-sensitive (LNCaP-C33) cells in a lymph node carcinoma of the prostate (LNCaP) cell model, suggesting that RPL32 may be involved in the development of human prostate cancer[25]. Xie found that RPL32 induces the progression of lung cancer via p53 degradation[26]. Zhao used a humanized mouse model to analyze the breast cancer genes controlled by the human mammary microenvironment, including RPL32[27]. The expression of RPL32 in circulating tumor cell (CTC) clusters is higher in breast cancer patients than in single CTCs, which have a higher metastatic potential[15]. Xu revealed the oncogenic activity of RPL32 in BC[19]. Nonetheless, the expression and functional role of RPL32 in TNBC NAC resistance are still unknown.

In the current study, we found that the RPL32 expression was increased in TNBC tissues compared with healthy tissues. This finding has been confirmed by other recent studies[19]. Xu found strong RPL32 staining in most BC specimens[19]. However, 30 (31.25%) of the patients in our research had significant RPL32 protein expression, which might be because our study only included triple-negative breast tumors. Most recently, a retrospective analysis of TNBC patients showed a pCR of 33% after taxane-based NAC[28]. The finding of our study was consistent with the previous one, with 30 patients (31%) who received anthracycline and taxane-based NAC, achieving pCR. Furthermore, we also found that the high RPL32 expression in TNBC was associated with lymph node metastasis, low MP grading, and adverse clinical outcomes. Silencing of RPL32 induces adriamycin chemosensitivity in TNBC cells. RPL32 upregulation promotes adriamycin chemoresistance in TNBC cells. In univariate analyses based upon pCR, we found a significant association between Ki-67, p53, and RPL32 expression and pCR. In multiple regression analysis, p53 expression and RPL32 expression were the independent factors associated with pCR. It provides further evidence of the RPL32 oncogenic activity in BC.

The principal mechanism of the RPL32-mediated adriamycin chemoresistance was investigated using western blot analysis. The RPL32 knockdown increased the apoptosis, as shown by increased expression of cleaved caspase-3 and Bax and decreased in Bcl2 expression, while RPL32 overexpression induced the opposite effects (Fig. 4A). AKT pathway imbalance has been detected in most solid tumors (including breast cancer)[29, 30]. In all breast cancers, the probability of AKT signaling pathway maladjustment was as high as 70%[31], The maladjusted AKT pathway regulates the cell cycle and promotes the proliferation of cancer cells, inhibits pro-apoptotic proteins, and promotes anti-apoptotic signals to prevent apoptosis[32]. In primary TNBC, the probability of AKT pathway mutation is 25% and increased in metastatic advanced triple-negative breast cancer[33]. Our study revealed the Akt pathway is essential for RPL32-induced chemoresistance.

These findings suggest that RPL32 may be responsible for TNBC tumorigenesis, progression, and impaired neoadjuvant chemotherapy response. It should also be emphasized that the present research has some limitations. First, no in vivo experiment was performed. Second, the clinical application of the RPL32 marker needs large-scale prospective experiments.

To our knowledge, this is the first analysis of RPL32 and Akt pathway activation in TNBC patients who received an anthracycline-based neoadjuvant chemotherapy regimen. Our findings implicated RPL32 as a predictor of pCR to Anthracycline-based NAC in TNBC patients. The research also found that RPL32's functional role in TNBC cells is dependent on AKT. Overall, we considered PRL32 as a potential target for reversing anthracycline resistance, but need further exploration.

Authors' Contributions

Huili Liu, Jianli Ma, Xue Fang, and Dabei Tang

Conceptualization: JM and HL; Data curation: DT; Formal analysis: HL and XF Funding acquisition: DT; Investigation: JM and DT; Methodology: JM , HL and XF, Project administration: JM and HL. Supervision: DT; Roles/Writing—original draft: DT; Writing-review and editing: All authors. All authors read and approved the final manuscript.

Funding

This study was funded by Youth Science Fund of National Natural Science Foundation of China (grant number: 81903063) and Haiyan Fund Project of Harbin Medical University Cancer Hospital (grant number: JJMS2022-07).

Institutional Review Board Statement: The study was approved by the Ethics Committee of Harbin Medical University Cancer Hospital.

Informed Consent Statement: All listed authors have participated in the study and approved the submitted manuscript.

Data Availability Statement: We hereby undertake that all data and materials are available.

Conflicts of Interest: The authors declare that they have no conflict of interest.

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Table 1. Correlations between RPL32 status and clinicopathologic factors in 96 breast cancer patients

Patient characteristics	n	RPL32 Negative	RPL32 Positive	Perason’s χ²	P value
Age (years)				2.746	0.124
≤ 50	44	34	10
>50	52	32	20
Tumor grade				0.582	0.612
II （25%）	24	15	9
III （75%）	72	51	21
Tumor size				2.818	0.447
T1	3	2	1
T2	68	50	18
T3	17	10	7
T4	8	4	4
Nodal status				5.476	0.027*
Negative	49	39	10
positive	47	27	20
Clinical stage				3.735	0.156
I	7	4	3
II	70	52	18
III	19	10	9
ECOG				3.142	0.137
0	80	58	22
1-2	16	8	8
Ki-67				1.247	0.372
≤14%	40	30	10
>14%	56	36	20
MP grade				4.990	0.029*
1+2+3	51	30	21
4	16	13	3
5	29	23	6
Chemotherapy response				8.705	0.033*
CR	37	28	9
PR	33	19	14
SD	20	17	3
PD	6	2	4

Table 2. Univariate and Multivariate logistics analysis of prognostic factors for TNBC patients from our hospital.

Clinical Variables	Univariate Analysis		Multivariate Analysis
Clinical Variables	HR (95%CI)	p Value	HR (95%CI)	p Value
RPL32 (high expression vs low expression)	0.237（0.074-0.757）	0.015*	0.254（0.076-0.850）	0.026*
Age,years （＞50 vs ≤50）	0.645（0.271-1.535）	0.321
Grade (2 vs 3)	0.538（0.206-1.408）	0.207
Nodal (negative vs positive)	0.509（0.246-1.418）	0.238
ECOG (0 vs 1~2)	0.692（0.203-2.356）	0.556
Ki67 (＞14% vs ≤14%)	0.382（0.158-0.927）	0.033*	0.428（0.176-1.097）	0.077
Size （T1、T2 vs T3、T4）	0.857（0.440-1.669）	0.650
Stage (I 、II vs III)	0.523（0.158-1.736）	0.290
P53 (negative vs positive)	0.282（0.102-0.780）	0.015*	0.313（0.108-0.904）	0.032*

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Expression of RPL32 predicts response to anthracycline-based neoadjuvant chemotherapy in triple-negative breast cancer

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