Oncogene: Only melatonin helps sleep?

There is amplification/overexpression of human epidermal growth factor HER2/ERBB2 in 20% of breast cancers
.
So far, HER2 positive (HER2+) is still the only biomarker with clinical application value in anti-HER2 targeted therapy.

Researchers have developed several HER2 targeted drugs, including monoclonal antibodies (trastuzumab and Pertuzumab).

There is amplification/overexpression of human epidermal growth factor HER2/ERBB2 in 20% of breast cancers

Nelatinib is a new generation of irreversible, receptor tyrosine kinase (HER1/EGFR, HER2 and HER4) small molecule inhibitors, and has recently been approved for the treatment of HER2+ breast cancer

.

Nelatinib is a new generation of irreversible, receptor tyrosine kinase (HER1/EGFR, HER2 and HER4) small molecule inhibitors, and has recently been approved for the treatment of HER2+ breast cancer

This study aims to clarify the potential effects of melatonin monotherapy and neratinib in the treatment of HER2+ breast cancer cell lines

1.

Melatonin therapy reproduces the effects of HER2 targeted drugs

1.

Firstly, the effect of melatonin on the survival rate of three HER2+ breast cancer cell lines was tested

2.

Melatonin reduces the stability of HER2 protein

2.

The effect of melatonin on the existing HER2 protein pool in HER2+ breast cancer cells was detected by the protein synthesis inhibitor epoxy amide (CHX).
The level of HER2 protein gradually decreased after CHX treatment, but was observed in melatonin-treated cells HER2 is more significantly degraded (Figure 2B, D)
.
This indicates that melatonin treatment collectively reduces the stability of HER2 protein

.

3.
Melatonin treatment destroys HER2 by promoting the endocytosis and lysosomal degradation of HER2

3.
Melatonin therapy destroys HER2 by promoting the endocytosis and lysosomal degradation of HER2 3.
Melatonin therapy destroys HER2 by promoting the endocytosis and lysosomal degradation of HER2

It is known that receptor stability is regulated by ubiquitin-proteasome and/or lysosome mechanisms
.
This study found that the proteasome inhibited by MG132 or Velcade further reduced the abundance of HER2 protein after melatonin treatment (Figure 3A, B)

.

It is known that receptor stability is regulated by ubiquitin-proteasome and/or lysosome mechanisms
.
This study found that the proteasome inhibited by MG132 or Velcade further reduced the abundance of HER2 protein after melatonin treatment (Figure 3A, B)
.
In contrast, the lysosomal inhibitor BAF restored the reduced HER2 levels (Figure 3C), indicating that melatonin may induce HER2 down-regulation mainly through endocytic lysosome degradation
.

Tests to detect the effect of melatonin on the cellular localization of HER2 protein in HER2+ breast cancer cells showed that HER2 protein was strongly expressed on the cell membrane of untreated HCC1954 cells and diffused in the cytoplasm, while the HER2 protein was strongly expressed in the cytoplasm of cells treated with melatonin.
The appearance of inner dots (Figure 3D) suggests that melatonin may induce HER2 subcellular trafficking
.
Consistent with this, flow cytometry analysis showed that melatonin significantly reduced the amount of HER2 on the cell surface in a dose-dependent manner (Figure 3E)
.
This indicates the potential role of melatonin in promoting the endocytosis of HER2
.

Tests to detect the effect of melatonin on the cellular localization of HER2 protein in HER2+ breast cancer cells showed that HER2 protein was strongly expressed on the cell membrane of untreated HCC1954 cells and diffused in the cytoplasm, while the HER2 protein was strongly expressed in the cytoplasm of cells treated with melatonin.
The appearance of inner dots (Figure 3D) suggests that melatonin may induce HER2 subcellular trafficking
.
Consistent with this, flow cytometry analysis showed that melatonin significantly reduced the amount of HER2 on the cell surface in a dose-dependent manner (Figure 3E)
.
This indicates the potential role of melatonin in promoting the endocytosis of HER2
.

Melatonin treatment resulted in a decrease in the expression of HSP90 and HSC70, and a compensatory increase in HSP70 expression (Figure 4A).
At the same time, the amount of co- immunoprecipitation of HSP90 and HER2 in melatonin-treated cells was also significantly reduced, suggesting that melatonin may induce HSP90 was separated from HER2 (Figure 4B)
.
Melatonin treatment resulted in increased levels of HER2 ubiquitination (Figure 4B)
.
Immunofluorescence staining analysis showed that melatonin treatment can clearly localize the internalized HER2 into punctate structures (Figure 4C)
.
In addition, in melatonin-treated HCC1954 cells, HER2 was co-localized with LAMP1 (advanced endosome/lysosomal marker) (Figure 4D)
.
In addition, the lysosomal inhibitor BAF can rescue the down-regulation of HER2 induced by melatonin (Figure 3C).
These data support the idea that melatonin destroys HER2 by promoting endocytosis and lysosomal degradation
.

Melatonin treatment resulted in a decrease in the expression of HSP90 and HSC70, and a compensatory increase in HSP70 expression (Figure 4A).
At the same time, the amount of co- immunoprecipitation of HSP90 and HER2 in melatonin-treated cells was also significantly reduced, suggesting that melatonin may induce HSP90 was separated from HER2 (Figure 4B)
.
Immune melatonin treatment resulted in increased levels of HER2 ubiquitination (Figure 4B)
.
Immunofluorescence staining analysis showed that melatonin treatment can clearly localize the internalized HER2 into punctate structures (Figure 4C)
.
In addition, in melatonin-treated HCC1954 cells, HER2 was co-localized with LAMP1 (advanced endosome/lysosomal marker) (Figure 4D)
.
In addition, the lysosomal inhibitor BAF can rescue the down-regulation of HER2 induced by melatonin (Figure 3C).
These data support the idea that melatonin destroys HER2 by promoting endocytosis and lysosomal degradation
.

4.
Melatonin enhances the cytotoxicity of Nelatinib in HER2+ breast cancer cells

4.
Melatonin enhances the cytotoxic effect of Nelatinib in HER2+ breast cancer cells 4.
Melatonin enhances the cytotoxic effect of Nelatinib in HER2+ breast cancer cells

Next, we will study whether melatonin can enhance the efficacy of the pan-HER kinase inhibitor Nelatinib in HER2+ breast cancer cells
.
After the synergistic treatment response was observed in all four breast cancer cell lines tested, the synergistic growth inhibitory effect of the drug combination was studied through the clonal survival test
.
Compared with monotherapy, the combined use of melatonin and neratinib induces significantly stronger growth inhibitory effects (Figure 5A, B)
.
In the test of the migration potential of HER2+ breast cancer , it was found that the gap between scratch areas in the melatonin+neratinib group was larger than that in the single-agent group (Figure 5C)
.
Although neratinib and melatonin each reduced HER2 protein abundance and phosphorylated HER2 signal to a lesser extent, the combination therapy produced a stronger inhibitory effect (Figure 5D)
.
Melatonin or neratinib alone produced moderate cytotoxicity, but the combined treatment significantly increased cell death (Figure 5E)
.
In summary, these data indicate that the combination of melatonin and neratinib has synergistic therapeutic activity on HER2+ breast cancer cells
.

Next, we will study whether melatonin can enhance the efficacy of the pan-HER kinase inhibitor Nelatinib in HER2+ breast cancer cells
.
After the synergistic treatment response was observed in all four breast cancer cell lines tested, the synergistic growth inhibitory effect of the drug combination was studied through the clonal survival test
.
Compared with monotherapy, the combined use of melatonin and neratinib induces significantly stronger growth inhibitory effects (Figure 5A, B)
.
After the synergistic treatment response was observed in all four breast cancer cell lines tested, the synergistic growth inhibitory effect of the drug combination was studied through the clonal survival test.
Compared with the monotherapy, the combination of melatonin and neratinib induced The growth inhibitory effect is significantly stronger (Figure 5A, B)
.
In the test of the migration potential of HER2+ breast cancer , it was found that the gap between scratch areas in the melatonin+neratinib group was larger than that in the single-agent group (Figure 5C)
.
Although neratinib and melatonin each reduced HER2 protein abundance and phosphorylated HER2 signal to a lesser extent, the combination therapy produced a stronger inhibitory effect (Figure 5D)
.
Melatonin or neratinib alone produced moderate cytotoxicity, but the combined treatment significantly increased cell death (Figure 5E)
.
In summary, these data indicate that the combination of melatonin and neratinib has synergistic therapeutic activity on HER2+ breast cancer cells
.
In the migration potential of HER2+ breast cancer , the gap between the scratched areas of the melatonin+neratinib group was larger than that in the single-drug group (Figure 5C)
.
Although neratinib and melatonin each reduced HER2 protein abundance and phosphorylated HER2 signal to a lesser extent, the combination therapy produced a stronger inhibitory effect (Figure 5D)
.
Melatonin or neratinib alone produced moderate cytotoxicity, but the combined treatment significantly increased cell death (Figure 5E)
.
In summary, these data indicate that the combination of melatonin and neratinib has synergistic therapeutic activity on HER2+ breast cancer cells
.

5.
The combined use of melatonin and neratinib can effectively block the growth of xenogeneic HCC1954 tumors

5.
The combined use of melatonin and neratinib can effectively block the growth of xenogeneic HCC1954 tumors 5.
The combined use of melatonin and neratinib can effectively block the growth of xenogeneic HCC1954 tumors 5.
Melatonin and Naira The combined use of tinib can effectively block the growth of xenogeneic HCC1954 tumors

Compared with the control group, melatonin and neratinib monotherapy significantly reduced the growth of HCC1954 xenograft tumors (Figure 6A)
.
It is worth noting that, compared with melatonin and neratinib alone, the combined use of melatonin and neratinib inhibited tumor growth (calculated based on average tumor volume and endpoint tumor weight) (Figure 6A) -D), and only the combination therapy achieved tumor reversal (
5/8) in a small number of tumors (Figure 6A, B) .
Consistent with the results of in vitro experiments found that melatonin destroys HER2 protein, mouse tumors treated with melatonin alone showed a significant reduction in HER2 protein abundance (Figure 6E)
.
We also found that the pan-HER kinase inhibitor Nelatinib reduced the abundance of HER2 protein and the HER2 signaling pathway (Figure 6E and Figure 5D)
.
In addition, the combination therapy did not produce obvious toxic effects during the whole treatment process
.
In summary, the combined use of melatonin and neratinib may improve the therapeutic effect of HER2 + breast cancer
.

Compared with the control group, melatonin and neratinib monotherapy significantly reduced the growth of HCC1954 xenograft tumors (Figure 6A)
.
It is worth noting that, compared with melatonin and neratinib alone, the combined use of melatonin and neratinib inhibited tumor growth (calculated based on average tumor volume and endpoint tumor weight) (Figure 6A) -D), and only the combination therapy achieved tumor reversal (
5/8) in a small number of tumors (Figure 6A, B) .
Consistent with the results of in vitro experiments found that melatonin destroys HER2 protein, mouse tumors treated with melatonin alone showed a significant reduction in HER2 protein abundance (Figure 6E)
.
We also found that the pan-HER kinase inhibitor Nelatinib reduced the abundance of HER2 protein and the HER2 signaling pathway (Figure 6E and Figure 5D)
.
The pan-HER kinase inhibitor neratinib In addition, the combination therapy did not produce significant toxic effects during the entire treatment process
.
In summary, the combined use of melatonin and neratinib may improve the therapeutic effect of HER2 + breast cancer
.
Neratinib

In summary, this study clarified the combined therapeutic effect of melatonin and neratinib in HER2+ breast cancer cells, and proved the destructive potential of melatonin on HER2
.
Both in vitro and in vivo experimental results show that melatonin significantly enhances the toxic effect of Nelatinib on HER2-positive breast cancer cells, even if these cells contain PIK3CA mutations and have a weaker response to HER2-targeted drugs
.
Future studies will test whether this drug combination can play a role in breast cancer that is resistant to her2 targeted drugs
.

In summary, this study clarified the combined therapeutic effect of melatonin and neratinib in HER2+ breast cancer cells, and proved the destructive potential of melatonin on HER2
.
This study clarified the combined therapeutic effect of melatonin and neratinib in HER2+ breast cancer cells, and proved the potential of melatonin to destroy HER2
.
Both in vitro and in vivo experimental results show that melatonin significantly enhances the toxic effect of Nelatinib on HER2-positive breast cancer cells, even if these cells contain PIK3CA mutations and have a weaker response to HER2-targeted drugs
.
Future studies will test whether this drug combination can play a role in breast cancer that is resistant to her2 targeted drugs
.
Both in vitro and in vivo experimental results show that melatonin significantly enhances the toxic effect of Nelatinib on HER2-positive breast cancer cells, even if these cells contain PIK3CA mutations and have a weaker response to HER2-targeted drugs
.
Future studies will test whether this drug combination can play a role in breast cancer that is resistant to her2 targeted drugs
.

In general, the results of the study indicate that melatonin + neratinib is expected to become a new HER2 dual blockade strategy for the treatment of HER2-positive breast cancer
.

In general, the results of the study indicate that melatonin + neratinib is expected to become a new HER2 dual blockade strategy for the treatment of HER2-positive breast cancer
.
In general, the results of the study indicate that melatonin + neratinib is expected to become a new HER2 dual blockade strategy for the treatment of HER2-positive breast cancer
.

 

Original source:

Z.
Liu et al.
Melatonin potentiates the cytotoxic effect of Neratinib in HER2+ breast cancer through promoting endocytosis and lysosomal degradation of HER2.

Oncogene; https://doi.
org/10.
1038/s41388-021-02015-w

 

 

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