HER-2 SMASH

doi:10.21203/rs.3.rs-4877263/v1

Download PDF

Research Article

HER-2 SMASH

https://doi.org/10.21203/rs.3.rs-4877263/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Purpose

Human epidermal growth factor-2 (HER-2) targeted drugs are used in only HER-2 overexpressed cancers. However, only a small portion of these cancer types are HER-2 overexpressed. In this study, we aimed to upregulate HER-2 receptors in MCF-7 breast cancer and HT-29 colon cancer cell cultures, which these cells are not HER-2 upregulated in natural status.

Methods

We used a 10-day non-cytotoxic lapatinib dose to upregulate HER-2 receptors. HER-2 levels of these cell lines were tested with ELISA and immunofluorescence tests before and after 10 days of lapatinib administration. After upregulation of HER-2, we administered trastuzumab, and T-DM1 to these cell lines to observe whether there is an increase in anticancer activity. We used a cell viability test (XTT) to show the cytotoxicity of trastuzumab and T-DM1. Also, we used ELISA and immunofluorescence for HER-2 pathway proteins to understand the mechanism of increased anti-cancer activity.

Results

We showed that administration of lapatinib for 10 days leads to overexpression of HER-2 receptors on both MCF-7 and HT-29 cells. A significant increase in the cytotoxicity of trastuzumab or T-DM1 was observed after 10 days of lapatinib administration.

Conclusion

We named this method the smash method, which is the volleyball term. In volleyball, the ball is raised while low and quickly hits the ground again, just like we do with the HER-2 receptor. The smash method can switch HER-2 negative or HER-2 low tumors into HER-2 overexpressed, iatrogenically. Thus, we can use her2-targeted therapies in all cancer patients instead of a small portion.

Her-2 overexpression

lapatinib

smash method

trastuzumab

Chronopharmacology is a branch of pharmacology studying the dependencies between the timing of drug administration and its effects on therapy (1). Through a chronopharmacological treatment approach, side effects can be reduced while increasing the effectiveness of drugs. Age, sex, body surface area, concomitant diseases, drug interactions, biological rhythms, and, genotyping can affect to phenotypic response to the administered drug. Drugs can change blood concentrations by altering each other's absorption and elimination, thereby changing their effectiveness and side effects. When one drug is administered before or after another, or when administered simultaneously, its pharmacokinetic and pharmacodynamic effects may differ (2). Chronopharmacological treatment plans are not frequently made in oncological treatments today. However, we think that chronopharmacological plans may be very important in cancer treatment, especially in targeted therapies. For example, lapatinib and trastuzumab, which are both targeted therapies, are used simultaneously. We do not know what the effectiveness of the treatment will be if lapatinib is started before trastuzumab treatment. Lapatinib is used every day after starting treatment, but we do not know whether intermittent use is more effective.

When a receptor is continuously blocked, there may be an increase in the expression of that receptor. This is a physiological response. HER-2 receptor is one of the most important targets in breast, colorectal, and gastric cancer treatment. When the HER-2 expression level of cancer tissue is evaluated pathologically by immunohistochemistry (IHC), it is named as negative (0), low (+ 1, + 2)) or positive (+ 3). Also if HER-2 IHC + 2 and in-situ hybridization (ISH) is amplified it is named as positive. In HER-2 positive breast cancer, it was shown that HER-2 expression levels have been associated with treatment benefits in the neoadjuvant and metastatic settings (3, 4). Also, the combination of trastuzumab and lapatinib in the treatment of metastatic colorectal cancer is effective in only the HER-2 positive group (5). HER-2 protein overexpression and/or HER2 gene amplification is found in about 20% of invasive breast cancers, 22% of gastric cancers, and 6% of colorectal cancers (9). Lapatinib, trastuzumab, and trastuzumab-emtansine (TDM-1) are all HER-2 targeted drugs that they used in HER-2 overexpressed tumors. Lapatinib is a potent reversible and selective inhibitor of the tyrosine kinase domains of epidermal growth factor receptor and HER-2 that exerts its action by competitive binding to the intracellular ATP-binding site of the receptor. It is used in advanced or metastatic HER-2 + breast cancer in combination with capecitabine (6). Trastuzumab is a HER-2 targeted drug. Trastuzumab improves overall survival (OS) and progression-free survival (PFS) in both early and advanced HER-2 positive breast cancer (7, 8). Trastuzumab combinations with other HER-2 targeted therapies like pertuzumab, tucatinib, lapatinib are used in HER-2 positive colorectal cancers (9). Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate, trastuzumab conjugated with the cytotoxic activity of the microtubule-inhibitory agent DM1. T-DM1 significantly prolonged PFS in patients with HER-2 positive breast cancer (10). Although HER-2 targeted agents have demonstrated significant outcomes in the treatment of HER-2 receptor-positive cancers, a considerable number of patients lack durable responses to treatment with HER-2 inhibition. It is a main problem for HER-2 targeted therapies. The other problem of HER-2 targeted problem is both primary and secondary resistance develops to HER-2 targeted therapies over time. The main mechanisms of resistance are HER-2 receptor mutations inducing impaired ligand binding, upregulation or downregulation of HER-2 receptors, activation of MET, HER3, or FGFR receptors, or activation of downstream signaling such as PI3K/AKT and MAPK (11).

Until recently, HER-2 targeted therapies were thought to be effective only in HER-2 overexpressed tumors, but DESTINY studies showed that another HER-2 targeted agent, trastuzumab-deruxtecan, was also effective in HER-2 -low tumors (12). In a clinical study, it was shown that HER-2 negative (IHC 0) breast cancers could be switched to HER-2 low (IHC + 1 or + 2) by receiving neoadjuvant one-month of tamoxifen or letrozole in patients with early-stage estrogen receptor-positive breast cancer (13). In another study, it was shown that when neoadjuvant chemotherapy was administered to patients with early-stage HER-2 negative breast cancer, 2.2% of them switched to HER-2 positive. It has been stated that this change may be due to the mechanism of resistance to chemotherapy or tumor heterogeneity (18). HER-2 negative tumors can become HER-2 positive as a mechanism of resistance to some tyrosine kinase inhibitors. In a study, it was shown, HER-2 negative gastric cancer became HER-2 positive after lapatinib therapy (14).

In this study, we planned to switch HER-2 negative cancer cells to HER-2 low or HER-2 positive by using lapatinib. We hypothesized that if we give low-dose lapatinib for a while, a resistance mechanism or tolerance may develop in tumor cells and increase Her-2 receptors. Our main goal is to sensitize cells to HER-2 targeted therapies such as trastuzumab or T-DM1 by increasing the HER-2 expression levels.

Cell Line and Reagents

Colon adenocarcinoma cell line HT-29 (ATCC HTB-38) and breast adenocarcinoma cell line MCF-7 (ATCC HTB-22) were purchased from the American Type Culture Collection. The cells were grown in RPMI-1640 medium (Capricorn Scientific) with 10% fetal bovine serum (Sigma-Aldrich), 1% antibiotic mixtures of penicillin, and streptomycin (Sigma-Aldrich) at 37°C in a 5% CO2 humidified incubator (15). Lapatinib was obtained from Novartis Pharma (Basel, Switzerland) and solubilized in DMSO (Final DMSO in the culture medium less than 1%). Trastuzumab (Herceptin ®) and Trastuzumab Emtansine (Kadcyla TM) were obtained from Sivas Numune Hospital.

Experimental Groups

The experimental design showed in Fig. 1. MCF-7 and HT-29 cells were divided into 9 groups. The control group was not treated. Cells in the LAP group were treated with 500 nM LAP (which was found maximum non-cytotoxic dose), T group with 10 µg/ml T, T-DM1 group with 100 µg/ml, LAP + T group with 500 nM LAP + 10 µg/ml T, LAP + T-DM1 group with 500 nM LAP + 100 µg/ml T-DM1 for 24 hours. Cells in the LAP10 group were treated with 500 nM LAP for 10 days. After 10 days of Lap treatment, 10 µg/ml T and 100 µg/ml T-DM1 were added to the cells in LAP10 + T and LAP10 + T-DM1 groups, respectively, and incubated for 1 day.

Cell Viability Test

Cell viability was determined using the XTT assay (Sartorius, Göttingen, Germany). Before the treatment, MCF-7 and HT-29 cells were seeded in 96-well plates at a density of 10*103 cells per well in 100-L RPMI-1640. The following day, nine cell groups were prepared to assess the cell viability of all groups. LAP, T, T-DM1, LAP + T, and LAP + T-DM1 groups were administered at the determined concentrations and incubated for 1 day. LAP was applied to other groups for 10 days, then T and T-DM1 were added and waited for 1 day. After incubation, the medium was removed, and phosphate-buffered saline was used to wash the wells twice. The plates were then incubated at 37°C for 4 hours after the last step of adding 100 µL of RPMI-1640 without phenol red and 50 µL of XTT labelling solution to each well. The absorbance values of the all plates were determined by ELISA microplate with a wavelength of 450 nm. Experiments were performed three times and cell viability was measured as percentages of the number of viable cells compared to untreated cells (control) (16).

Preparation of Cell Homogenates

Cells in each group were collected in sterile tubes. The supernatant was removed after centrifugation at 2000 rpm for approximately 10 minutes. Cells in the tubes were suspended by diluting the cell suspension with PBS (pH: 7.4) to a cell concentration of approximately 1 million/ml. The cells were damaged by repeated freeze-thaw cycles that allowed internal components to escape. Then the cells were then centrifuged for 10 minutes at 4°C using at 4000 rpm. The supernatants obtained were then collected and subjected to biochemical analysis. Total protein levels in the samples were determined using the Bradford protein analysis kit (Merck Millipore, Darmstadt, Germany) (17).

The measurement of HER-2, EGFR, ERK-1, ERK-2, mTOR, BAD and Caspase-3 quantities

The human ELISA kits of HER-2 (8-oxo-dG) (BT Lab, catalogue #E0224Hu), epidermal growth factor receptor (EGFR) (BT Lab, catalogue #E0313Hu), extracellular signal-regulated kinase 1 (ERK-1) (BT Lab, catalogue #E4263Hu), extracellular signal-regulated kinase 2 (ERK-2) (BT Lab, catalogue #E4264Hu), mammalian target of rapamycin (mTOR) (BT Lab, catalogue #E3693Hu), Bcl2 antagonist of cell death (BAD) (BT Lab, catalogue #E4102Hu) and caspase 3 (BTLab, catalogue # E4804Hu) were used to examine the quantities of HER-2, EGFR, ERK-1, ERK-2, mTOR, BAD and caspase 3 in LAP, T, T-DM1-treated and untreated MCF-7, and HT-29 cells. The ELISA assay protocol was performed according to the procedures written in the user manual. At the end of the procedure, in an ELISA reader (Thermo Fisher Scientific, Altrincham, U.K.), the absorbance values resulting from the colour change of control, untreated, and treated cell samples were measured at 450 nm (18). Then, following the manufacturer's directions, the quantities of HER-2, EGFR, ERK-1, ERK-2, mTOR, BAD, and caspase-3 in cell lysates were determined.

Immunofluorescent Microscope Analysis of HER-2 and P21

For p21 mouse monoclonal antibody and HER2 immunofluorescence staining, HT29 and MCF7 cells were grown on sterile coverslips in 6-well culture dishes. The cells were then treated with drug treatment. After treatment, the cells were purified from the medium and washed with phosphate-buffered saline (PBS) (Sigma Aldrich, Germany). Washed cells were fixed in paraformaldehyde (for p21 antibody) (pH 7.4) (Merk, Germany) and %10 buffered neutral formalin (for HER2 antibody) for 10 min. At room temperature. After fixation, wash it 3 times with cold PBS. Subsequently, the sodium citrate buffer (pH 6.5) heated at 90℃ was treated for 10 minutes (Sigma Aldrich, Germany) to reveal the tissue antigen. Cells were washed 2 times with PBS for 5 min. For permeability, cells were incubated in 0.1% TritonTM X-100 (Sigma Aldrich, Germany) solution for 10 min. The PBS was then washed for 5 minutes. After washing, Ultra V Block (Thermo Scientific, PBQ180830, USA) was dropped and left at room temperature for 30 min. to avoid non-specific binding. Cells were then incubated with Mause Monoclonal P21 (Thermo Scientific, USA) and anti-HER2/neu 4B5 rabbit monoclonal (Ventana) primary antibody at 36℃ in the dark. It was kept at + 4℃ overnight. At the end of the period, it was washed twice with PBS for 5 min. A secondary antibody to the Goat AntiMouse IgG H & L antibody (Alexa Fluor® 488) (ab150113) (Abcam, USA) was used for the p21 primer antibody. A secondary antibody to Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) (Abcam, USA) was used for the HER2 primer antibody. Secondary antibodies were diluted to 1:200 with antibody diluent reagent (Invitrogen, USA) and applied to cells for 1 h. in a humidity chamber. It was then stained with 0.1 µg/mL 4'6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma Aldrich, Germany), and after washing with PBS, the coverslips from the culture dishes were removed and inverted on the slide. Fluorescence microscopy (Olympus BX51, Japan) was studied using filters suitable for fluorescence examination followed by a recording. In the examination, the intensity of the primary antibody positivity in the cells under the fluorescence microscope was evaluated semiquantitatively as absent (-), mild (1), moderate (2), and severe (3).

Immunofluorescent Microscope Analysis of EGFR, Erk1/2 and mTOR

Cells were fixed with methanol for 5 minutes at -20º C and washed with PBS. Then, it was left for 15 minutes with PBS containing 0.1% Triton X-100 at room temperature. After washing, it was incubated with PBS containing 2% BSA for 60 minutes at room temperature. After further washing, monoclonal anti-ERK1/2 (Santa Cruz, Catalog no. sc-514302), monoclonal anti-mTOR (Santa Cruz, Catalog no. sc-517464) and monoclonal anti-EGFR (Santa Cruz, Catalog no. sc-51) -373746) was incubated overnight at + 4ºC with primary antibodies at a dilution of 1/100. Cells washed with PBS were incubated with goat anti-mouse FITC secondary antibody (Jackson ImmunoResearch, Catalog no. 115-095-003) at a dilution of 1/50, compatible with the primary antibodies used, for 1 hour at room temperature in the dark. Finally, 4′,6-diamidino-2-phenylindole (DAPI) was dropped onto the washed cells and examined under a fluorescence microscope (Zeiss Axio). In the examination, the intensity of fluorescence positivity in the cells was evaluated with the Fiji Image J program.

Statistical analysis

For cell viability and ELISA test results Statistical analysis was performed by post hoc Tukey test, comparing cells treated with Lapatinib, Trastuzumab, trastuzumab emtansine, and different combinations with a control group using one-way ANOVA and SPSS Version 23.0 for Windows.

For immunofluorescent microscope results statistical analysis was performed by post hoc Tukey test, comparing cells treated with Lapatinib, Trastuzumab, trastuzumab emtansine, and different combinations with a control group using one-way ANOVA and SPSS Version 23.0 for Windows.

Effect of LAP, T, T-DM1, and Combination Treatments on MCF-7 and HT-29 Cell Viability

This study investigated the effects of LAP, T, T-DM1, and combination treatments on cell survival in both cell lines. We showed that 10 days of lapatinib administration in MCF-7 and HT-29 cells increased cell viability compared to the control group (p < 0.001; Fig. 2). In addition, trastuzumab and trastuzumab emtansine administered to cells after 10 days of lapatinib administration decreased cell survival in both cell lines (p < 0.001; Fig. 2).

Effect of LAP, T, T-DM1, and Combination Treatments on HER-2 Levels in MCF-7 and HT-29 Cell Line

ELISA was used to evaluate the expression of HER-2, the protein associated with the growth signal pathway, in MCF-7 and HT-29 cells. Lapatinib administration for 10 days significantly increased the expression of HER-2 protein in both cell lines. Also, HER-2 overexpression after 10-day lapatinib administration to both cell lines were showed in immunofluorescence tests (p < 0.001). After 10 days of lapatinib administration, trastuzumab and trastuzumab emtansine administration significantly decreased the expression of HER-2 protein (p < 0.01; Figs. 3).

Effect of LAP, T, T-DM1, and Combination Treatments on EGFR, ERK-1, ERK-2, and, mTOR Levels in MCF-7 and HT-29 Cell Line

ELISA was used to determine EGFR, ERK-1, ERK-2, and mTOR levels, which are proteins associated with the growth signalling pathway in MCF-7 and HT-29 cells. Administration of lapatinib for 10 days significantly increased the level of EGFR, ERK-1, ERK-2, and mTOR proteins in MCF-7 and HT-29 cell lines (p < 0.001). After 10 days of lapatinib administration, 24-hour treatment with trastuzumab and trastuzumab emtansine significantly reduced the levels of EGFR, ERK-1, ERK-2, and mTOR proteins (p < 0.01). In the other groups, EGFR, ERK-1, ERK-2, and mTOR levels were not affected (p > 0.05; Fig. 4).

The effect of LAP, T, T-DM1, and combination treatments on BAD and caspase-3 quantities in MCF-7 and HT-29 cells

ELISA was used to assess the expression of apoptosis-related proteins in MCF-7 and HT-29 cells, such as BAD and Caspase-3. After 10 days of lapatinib administration, 24-hour treatment with trastuzumab and trastuzumab emtansine significantly increased BAD (p < 0.05) and Caspase-3 (p < 0.001). However, trastuzumab and trastuzumab emtansine alone did not affect the amount of BAD and Caspase-3 (p > 0.05) (Fig. 5).

Analysis of HER-2 and P21 Immunofluorescence Tests Results

In the immunofluorescence staining performed with Anti HER2 and P21 antibodies on MCF7 and HT29 cell lines, a statistically significant difference was detected between the groups (Table 1, and 2, p < 0.05). In the HT29 cell line, the most intensely fluorescent group in terms of fluorescence positivity of Anti HER2 antibody is L500 (10 days). In the HT29 cell line, the groups with the most severe fluorescence positivity for the P21 antibody are the T + L500 (10 days) and then the T-DM1 + L500 (10 days) groups, respectively. Moderate positivity was observed in the T-DM1 + L500 (day 1) group (p < 0.05). P21 is the protein that stops the cell cycle in the g1 phase and causes cells to undergo apoptosis. In the HT29 cell line, the groups with the most severe fluorescence positivity for the P21 antibody are the T + L500 (10 days) and then the T-DM1 + L500 (10 days) groups, respectively. While moderate positivity was observed in theT-DM1, T-DM1 + L500 (1 day), and L500 (10 day) groups, the lowest positivity was observed in the control, L500 (1 day), T (1 day) and T + L500 (1 day) groups.

Table 1

Statistical differences in immunofluorescence staining of Anti-Her2 and P21 antibodies in the MCF-7 cell line. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared to the control groups.
Groups	Anti HER2	P21
Control	1,2 ± 0.40	0.2 ± 0.40
L500 (1 day)	2,2 ± 0.40 *	0.4 ± 0.60
T (1 day)	1.8 ± 0.40	0.6 ± 0.40
T + L500(1 day)	1.8 ± 0.40	0.8 ± 0.20
T-DM1 (1 day)	1.7 ± 0.30	1.2 ± 0.40
T-DM1 + L500 (1 day)	1.8 ± 0.20	1.8 ± 0.20*
L500(10 day)	2.8 ± 0.20 ***	1.4 ± 0.20
T + L500(10 day)	0.8 ± 0.20	2.8 ± 0.20***
T-DM1 + L500(10 day)	0.6 ± 0.20	3 ± 0***

Table 2

Statistical differences in immunofluorescence staining of Anti-HER2 and P21 antibody localization in the HT-29 cell line. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared to the control groups.
Groups	Anti HER2	P21
Control	1.2 ± 0.20	0.2 ± 0.20
L500 (1 day)	2.0 ± 0.40	0.2 ± 0.20
T (1 day)	1.8 ± 0.20	0.4 ± 0.40
T + L500(1.day)	1.6 ± 0.20	0.6 ± 0.40
T-DM1 (1 day)	1.4 ± 0.60	1 ± 0.40
T-DM1 + L500 (1 day)	1.6 ± 0.40	1.4 ± 0.60*
L500(10 day)	2.6 ± 0.40**	0.6 ± 0.20
T + L500(10 day)	0.6 ± 0.40	2.6 ± 0.40***
T-DM1 + L500(10 day)	0.4 ± 0.20	2.8 ± 0.20***

Analysis of EGFR, Erk1/2, and mTOR Immunofluorescence Tests Results

A statistically significant difference was detected between the groups in immunofluorescence staining with ERK 1/2, mTOR, and EGFR (Table 3, and 4, p < 0.001). ERK 1/2, mTOR and EGFR fluorescence positivity was most severe in the Control, L500 (1 day), T (1 day), and L500 (10 day) groups. While positivity was found to be moderate in T + L500 (1 day), T-DM1 (1 day) and T-DM1 + L500 (1 day) from other application groups, T + L500 (10th day) and T-DM1 + L500 (1 day) On the 10th day, positivity was detected at a mild level (Fig. 15–17).

Table 3

Statistical differences in ERK 1/2, mTOR and EGFR immunofluorescence stainings (MCF-7 cell line). (*,**,*** indicate difference between groups, p < 0.001).
Groups	ERK1/2	mTOR	EGFR
Control	56,16 ± 5,35^*	49,31 ± 9,26^*	53,27 ± 6,42^*
L500 (1 day)	53,26 ± 9,14^*	51,34 ± 5,45^*	55,82 ± 3,65^*
T (1 day)	54,87 ± 7,65^*	52,60 ± 3,06^*	55,02 ± 4,28^*
T + L500(1 day)	43,20 ± 4,08^**	44,71 ± 8,16^**	40,12 ± 4,09^**
T-DM1 (1 day)	41,54 ± 3,91^**	42,32 ± 6,04^**	39,50 ± 9,36^**
T-DM1 + L500 (1 day)	44,19 ± 3,05^**	42,04 ± 9,62^**	42,56 ± 3,51^**
L500(10 day)	56,03 ± 2,17^*	52,18 ± 3,48^*	54,30 ± 4,72^*
T + L500(10 day)	37,31 ± 7,80^***	39,64 ± 6,08^***	37,31 ± 7,80^***
T-DM1 + L500(10 day)	39,23 ± 8,42^***	41,72 ± 4,06^***	35,48 ± 1,24^***

Table 4

Statistical differences in ERK 1/2, mTOR and EGFR immunofluorescence stainings (HT-29 cells). (*,**,*** indicate difference between groups, p < 0.001).
	ERK1/2	mTOR	EGFR
Control	52,31 ± 6,04^*	54,18 ± 5,12^*	50,34 ± 3,96^*
L500 (1.day)	50,08 ± 3,41^*	55,06 ± 4,98^*	48,26 ± 9,32^*
T (1 day)	49,20 ± 4,60^*	53,59 ± 7,04^*	51,82 ± 3,68^*
T + L500 (1 day)	41,56 ± 6,78^**	44,71 ± 8,16^**	42,30 ± 8,24^**
T-DM1 (1 day)	43,37 ± 7,04^**	42,32 ± 6,04^**	41,02 ± 6,18^**
T-DM1 + L500 (1 day)	41,02 ± 4,82^**	42,04 ± 9,62^**	43,14 ± 2,40^**
L500 (10 day)	49,80 ± 9,56^*	52,45 ± 4,17^*	49,62 ± 6,34^*
T + L500 (10 day)	35,32 ± 3,62^***	34,02 ± 2,78^***	33,41 ± 2,04^***
T-DM1 + L500 (10 day)	36,12 ± 6,04^***	36,02 ± 5,82^***	34,21 ± 5,30^***

In volleyball, when a player raises the ball while it is close to the ground and the other player quickly hits the ball toward the opponent's court, it is called a smash (19). In this study, we considered the HER-2 receptor instead of the ball, 10-day low-dose lapatinib instead of the player who raises the ball, and trastuzumab or T-DM1 instead of the player who smashes the ball. This is why we named the method HER-2 smash. Such a method is not yet used in oncology practice.

This is the first study showing HER-2 could be overexpressed with low-dose lapatinib. This means we can modify the receptor levels on the tumor cell with drugs. By changing the receptor levels in cancerous tissues, we can make them candidates for targeted treatments. In our study, we administered lapatinib for 10 days to MCF-7 and HT-29 cells, which were HER-2 negative initially, both cells became HER-2 positive. We also showed HER-2, Erk1/2, mTOR, and EGFR protein levels increased when 10 days of lapatinib were administered by ELISA and immunofluorescence staining tests. In other words, not only HER-2 receptor levels but also post-receptor proteins increase. When a receptor is blocked, there will be an increase in that receptor’s density on the cell surface. It is a physiological response. This response is called tolerance. By tolerance mechanism, even if the receptor is blocked, the cells continue to receive the stimulus they need. (20). Based on this mechanism, we started our study with the question of whether we could increase the HER-2 expression intensity if we blocked the HER-2 receptors. When we blocked the HER-2 receptor intracellular pathway, the response of the cells was to increase the expression of the HER-2 receptor. The reason why we applied lapatinib for 10 days was that in previous studies, morphine or beta-adrenergic receptor blocking generally caused an increase in expression on the 10th day (21). The purpose of applying the non-cytotoxic maximum dose of lapatinib was to increase HER-2 expression without killing the cells. Interestingly, in this study, there was an increase in the viability of both HT-29 and MCF-7 cells administered lapatinib at a non-cytotoxic dose for 10 days, a decrease in apoptosis, and an increase in HER-2 receptor and post-receptor proteins.

The status of the HER-2 expression in cancer is not static and may change among the primary tumor, lymph node metastases, and distant metastases. This HER-2 status change can be a consequence of the natural evolution of the tumor or resistance to therapy (22). Lapatinib is a HER-2 pathway-specific TKI, it blocks the intracellular domain of the HER-2 receptor. HER2 signalling is mainly transduced by the phosphatidylinositol 3-kinase (PI3K)/Akt and the Erk1-2/MAPK survival pathways. Resistance mechanisms against lapatinib can be grouped under 3 headings: activation of compensatory pathways, mutation of HER-2 receptor domains, and amplification of some genes (23). However, all of these resistance mechanisms have been identified in HER-2-positive cancers. Since lapatinib was found to be ineffective in all studies of HER-2 negative cancers, resistance mechanisms were not investigated. However, in a study, it was shown that lapatinib received HER-2 negative cancers would become HER-2 positive over time. Although they attribute this to tumor heterogeneity, the exact reason is unknown. In the same study, although HER-2 amplification was correlated with response to treatment in gastric cancer patients, HER-2 amplification disappeared while lapatinib treatment continued. Then HER-2 was amplified again when lapatinib resistance developed (14). Conversely, another study also demonstrated that HER-2 expression can change after trastuzumab-based chemotherapy in patients with advanced HER-2-positive gastric cancer (24).

In this study, our aim in using both HT-29 and MCF-7 cells was to show that the smash mechanism can also work in different types of cancer. We showed if HER-2 receptor expression level increases, the cytotoxic effects of trastuzumab and T-DM1 increase. We also showed that the increase in cytotoxic effect is due to the increase in apoptosis (p21 protein) and decrease of HER-2 pathway proteins (HER-2, Erk1/2, mTOR, and EGFR). In both ELISA and immunofluorescence tests, a significant increase in HER-2 pathway proteins was observed in the cells administered lapatinib for 10 days, and after the administration of trastuzumab or T-DM1, HER-2 pathway proteins were significantly reduced in the same cells. In the Destiny-breast 1 study, it was shown that as the HER-2 expression level increased, the anti-cancer effectiveness of trastuzumab-deruxtecan increased (12). For this reason, we think that being able to iatrogenically increase the expression of the HER-2 receptor is very important in cancer treatment.

We think that if this smash method is successful, it can be used in many oncological treatments. However, the smash method may have various handicaps. May cause rapid growth of the tumor, and may not work in vitro. It may increase the side effects of HER-2 targeted therapies by increasing the HER-2 receptor in healthy tissues in vitro. First of all, the smash method needs to be tested with animal experiments.

This is a cell line study. There is no need for the ethics committee's approval for cell line studies in our country.

Author Contribution

C.A. Wrote the main manuscript text, established the study hypothesis and manage the studyA.Ö. She performed cell culture, XTT tests and ELISA tests and wrote the material and methods section of the study, created the figuresF. Y. He performed cell culture, XTT tests and ELISA tests and wrote the material and methods section of the studyZ. De. Ş. İ. Performed the immunofluorescence tests and wrote the material and methods section of the studyM. Ö. Performed the immunofluorescence tests and wrote the material and methods section of the study, created the tables and figures.B. B. L. Performed the immunofluorescence tests and wrote the material and methods section of the study, created the tables and figures.A. A. Consultant of the study.All authors reviewed the manuscript.There is no reproduced images. All images produced by this manuscript authors.This is a cell line study. There is no need for ethics committee approval in our country.

Acknowledgement

The authors thank Sivas Cumhuriyet University Medical Faculty Research Center (CUTFAM) and the Turkish Society of Medical Oncology (TTOD) for providing the necessary facilities to conduct this study.

Andrys-Wawrzyniak I, Jabłecka A (2008) Chronobiology, chronopharmacology on medicine (part I). Farm Współ 1:94–108
Lesko LJ, Schmidt S (2012) Individualization of drug therapy: history, present state, and opportunities for the future. Clin Pharmacol Ther 92(4):458–466
Gianni L, Pienkowski T, Im Y-H, Tseng L-M, Liu M-C, Lluch A et al (2015) Five-year analysis of the phase II NeoSphere trial evaluating four cycles of neoadjuvant docetaxel (D) and/or trastuzumab (T) and/or pertuzumab (P). American Society of Clinical Oncology
Eiermann W, Group IHS (2001) Trastuzumab combined with chemotherapy for the treatment of HER2-positive metastatic breast cancer: pivotal trial data. Ann Oncol 12:S57–S62
Tosi F, Sartore-Bianchi A, Lonardi S, Amatu A, Leone F, Ghezzi S et al (2020) Long-term clinical outcome of trastuzumab and lapatinib for HER2-positive metastatic colorectal cancer. Clin Colorectal Cancer 19(4):256–262 e2
Opdam FL, Guchelaar HJ, Beijnen JH, Schellens JH (2012) Lapatinib for advanced or metastatic breast cancer. Oncologist 17(4):536–542
Lee SW, Kamaruzaman HF, Sabirin J (2018) Trastuzumab as An Adjuvant Therapy for Early Breast Cancer. J Global Oncol 4(Supplement 2):70s–s
McKeage K, Perry CM (2002) Trastuzumab: a review of its use in the treatment of metastatic breast cancer overexpressing HER2. Drugs 62(1):209–243
Bando H, Ohtsu A, Yoshino T (2023) Therapeutic landscape and future direction of metastatic colorectal cancer. Nat Rev Gastroenterol Hepatol 20(5):306–322
Verma S, Miles D, Gianni L, Krop IE, Welslau M, Baselga J et al (2012) Trastuzumab Emtansine for HER2-Positive Advanced Breast Cancer. N Engl J Med 367(19):1783–1791
Blangé D, Stroes CI, Derks S, Bijlsma MF, van Laarhoven HWM (2022) Resistance mechanisms to HER2-targeted therapy in gastroesophageal adenocarcinoma: A systematic review. Cancer Treat Rev 108:102418
Modi S (2021) Trastuzumab deruxtecan in previously treated HER2-positive metastatic breast cancer: plain language summary of the DESTINY-Breast01 study. Future Oncol 17(26):3415–3424
Chaudhary LN, Jorns J, Sun Y, Cheng YC, Kamaraju S, Burfeind J et al (2023) Frequent Upregulation Of HER2 Protein In Hormone Receptor-Positive HER2-Negative Breast Cancer After Short-Term Neoadjuvant Endocrine Therapy. Res Sq
Kim ST, Banks KC, Pectasides E, Kim SY, Kim K, Lanman RB et al (2018) Impact of genomic alterations on lapatinib treatment outcome and cell-free genomic landscape during HER2 therapy in HER2 + gastric cancer patients. Ann Oncol 29(4):1037–1048
Ataseven D, Öztürk A, Özkaraca M, Joha Z (2023) Anticancer activity of lycopene in HT-29 colon cancer cell line. Med Oncol 40(5):127
Zıad J, Yulak F, Öztürk A, Şahin B, YILDIRIM Ş (2021) The anticancer effect of cannabinoid 2 agonist L-759,633 on C6 and SH-SY5Y cell lines. Turkish J Sci Health 2(3):6–13
Filiz AK, Öztürk A (2021) The effect of carbamazepine against glutamate-induced cytotoxicity in the C6 cell line. Int J Sci Technological Res 7(8):67–76
Ergül M, Taşkıran AŞ (2021) Thiamine protects glioblastoma cells against glutamate toxicity by suppressing oxidative/endoplasmic reticulum stress. Chem Pharm Bull 69(9):832–839
Yusfi H (ed) (2021) Development of learning technique smash volleyball games. 4th Sriwijaya University Learning and Education International Conference (SULE-IC 2020); : Atlantis Press
Golf S, Hansson V (1986) Effects of beta blocking agents on the density of beta adrenoceptors and adenylate cyclase response in human myocardium: intrinsic sympathomimetic activity favours receptor upregulation. Cardiovascular Res 20(9):637–644
Rahban M, Danyali S, Zaringhalam J, Manaheji H (2022) Pharmacological blockade of neurokinin1 receptor restricts morphine-induced tolerance and hyperalgesia in the rat. Scand J Pain 22(1):193–203
Carlsson J, Nordgren H, Sjöström J, Wester K, Villman K, Bengtsson NO et al (2004) HER2 expression in breast cancer primary tumours and corresponding metastases. Original data and literature review. Br J Cancer 90(12):2344–2348
D'Amato V, Raimondo L, Formisano L, Giuliano M, De Placido S, Rosa R et al (2015) Mechanisms of lapatinib resistance in HER2-driven breast cancer. Cancer Treat Rev 41(10):877–883
Kijima T, Arigami T, Uenosono Y, Hiraki T, Yanagita S, Matsushita D et al (2020) Comparison of HER2 status before and after trastuzumab-based chemotherapy in patients with advanced gastric cancer. Anticancer Res 40(1):75–80

No competing interests reported.

Download PDF

Reviews received at journal
25 Sep, 2024
Reviewers agreed at journal
05 Sep, 2024
Reviewers invited by journal
03 Sep, 2024
Editor assigned by journal
08 Aug, 2024
Submission checks completed at journal
08 Aug, 2024
First submitted to journal
07 Aug, 2024

You are reading this latest preprint version

HER-2 SMASH

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Material and Method

Cell Line and Reagents

Experimental Groups

Cell Viability Test

Preparation of Cell Homogenates

The measurement of HER-2, EGFR, ERK-1, ERK-2, mTOR, BAD and Caspase-3 quantities

Immunofluorescent Microscope Analysis of HER-2 and P21

Immunofluorescent Microscope Analysis of EGFR, Erk1/2 and mTOR

Statistical analysis

Results

Effect of LAP, T, T-DM1, and Combination Treatments on MCF-7 and HT-29 Cell Viability

Analysis of HER-2 and P21 Immunofluorescence Tests Results

Analysis of EGFR, Erk1/2, and mTOR Immunofluorescence Tests Results

Discussion

Declarations

Author Contribution

Acknowledgement

References

Additional Declarations

Status:

Version 1