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Health Strategy.

Specific therapy for breast cancer.

The diversity of individual starting conditions dictates differences in treatment strategies and protocols. So, in some cases, the first step is surgical removal of the tumor, and in others, prior chemotherapy before surgery (neoadjuvant chemotherapy). In some cases, it is necessary to deal with the primary tumor, in others – with metastases. Other individual characteristics may be cancer subtype, menopausal status, gene predisposition, age, comorbidities, etc.

Breast cancer treatment protocols largely depend on its receptor subtype, which is characterized by the status of cellular receptors for estrogen (ERα), progesterone (PR), and human epidermal growth factor receptor type 2 (HER2). The presence of one or more of these receptors offers hope that treatments targeting these pathways will be effective. However, different chemotherapeutic solutions may be required for an effective therapeutic response for each cancer subtype.

Anti-HER2 therapy is only effective in tumors with HER2 up/overexpression; anti-estrogen therapy is effective in estrogen-responsive tumors; and PARP inhibitors are only effective in tumors with a BRCA * mutation. Expressive therapeutic responses are observed in B-RAF mutant melanoma using a B-RAF inhibitor *, and in lung cancer containing ALK mutations using an ALK inhibitor *.

In ER-positive breast cancer subtypes, selective estrogen receptor modulators (SERMs) and other methods of managing estrogen levels are used. In addition to tamoxifen and other similar drugs widely practiced for these purposes, there are other natural modulators that, although less effective, have weaker or zero side effects.

In ER-negative subtypes, estrogen reduction is likely to be ineffective. However, these subtypes may be sensitive to molecules critical for the growth and progression of ER-independent tumors. Such as HER receptor family inhibitors, cyclooxygenase-2 (COX-2) inhibitors * *, metformin and melatonin *, retinoids *, statins *, vitamin D * * and ω-3 fatty acids *, some other natural substances * including epigallocatechin gallate (EGCG) *, curcumin *, luteolin *, carotenoids *, resveratrol * * *, berberine * *, I3C *, ellagic acid *.

Another problem associated with targeted therapy is resistance, both primary and acquired. Acquired drug resistance eventually develops in most patients at an advanced stage of the disease *.
Addition, activation of compensatory pathways can save cells from the effects of blocking a single target or signaling pathway. For example, inhibition of the PI3K/Akt/mTOR pathway causes compensatory activation of several survival pathways, including the expression and phosphorylation of multiple receptor tyrosine kinases (RTKs) *.

Marker profiles of breast cancer Open in new window

In a wide range of tumor types, AKT inhibition induces signaling through a set of RTKs, including HER3, IGF-1R, and the insulin receptor; this may reduce their antitumor activity. In this situation, the combined inhibition of Akt and HER kinase activity is more effective than either alone *. Similarly, inhibition of PI3K signaling results in HER2 activation *, which is a major cause of blocking HER2 signaling in addition to PI3K.

In HER2-positive tumors, several mechanisms have also been identified by which a tumor stops responding to the therapy it originally did. These include reduction or loss of target expression as a result of continuous therapy *; activation of mutations associated with the therapeutic target * and activation of additional mechanisms that promote cell proliferation *. The presence of primary or secondary resistance also determines the characteristics of the treatment strategy.

The next conclusion that follows from the above is that it is worth using the widest possible combination treatment that can block the detours that allow cancer cells to develop resistance and elude targeted therapy. To use all the paths that are available to us. All available chances.

ER/PR-positive breast cancer.

Luminal ER-positive and PR-positive breast tumors account for approximately three-quarters of all breast cancers.

The luminal subtype A is an indolent disease, and is usually treated with hormonal therapy that either antagonizes/destroys the ER or inhibits aromatase, an enzyme that is critically involved in estradiol biosynthesis.

Luminal subtype B develops more actively than subtype A. Although it is predominantly HER2-negative, some of its variations express HER2 while retaining other characteristics of the HER2-negative luminal subtype. Proliferation markers such as cyclin B1, Ki-67 and growth factor proliferative signals are highly expressed here * *.

Luminal ER/PR-positive tumors are considered a typical example of cancer that responds to targeted therapy. Therapy aimed at reducing estrogen activity remains key to the treatment of this disease. A commonly used ER modulator, tamoxifen, reduces the incidence of breast cancer by about 40-50% and improves survival in patients with early and advanced breast cancer *. Further enhancements to this therapy * * are provided by aromatase inhibitors (Ai) * as well as fulvestrant, an estrogen receptor disruptor *.

However, despite short-term efficacy, this therapy can lead to the development of resistance to therapeutic drugs and the return of the disease. Despite continuous expression of ER during tumor recurrence, up to 50% of patients with HR-positive primary breast cancer who develop metastasis do not respond to hormonal treatment (primary resistance), and the remainder will eventually relapse despite initial success (secondary resistance) *.

AGTR1 (angiotensin II type I receptor) is markedly overexpressed in 10-20% of ER-positive, HER2-negative breast cancers. In these cases, losartan reduced the growth of the grafted mammary tumor in animals by 30% *, but clinical studies have not yet been conducted. A side effect of losartan is a decrease in blood pressure, which may be contraindicated in some cases.

Despite the possible success of additional (natural) substances, it remains unclear their long-term effectiveness, the impact on the development of resistance, patient survival and the possibility of regression of the disease.

HER2-overexpressing breast cancer.

The HER2 subtype, which accounts for about 15-20% of all breast cancers, is ER-negative. That is, in relation to it, the strategies discussed above may be ineffective. In patients with HER2+ breast tumors, the cancer cells produce too much of the human epidermal growth factor receptor 2 (HER2) transmembrane receptor protein. It is a receptor tyrosine kinase that is involved in signaling pathways that stimulate tumor cell proliferation. Overexpression of HER2 is associated with aggressive tumors, high rates of metastasis and recurrence, poor prognosis and treatment limitation caused by the acquisition of drug resistance (particularly to tamoxifen) * *.

Targeted therapy.

At the same time, HER2-positive cancer subtype responds to targeted therapy directed at the human epidermal growth factor receptor type 2 (HER2). In targeted therapy of HER2 tumors, monoclonal bodies are used – special antibodies that can attach to HER2 receptors of cancer cells and deactivate them. Blocking HER2 receptors on the surface of cancer cells means that cells do not receive the signal that encourages them to grow. Thus, the self-stimulation of cancer cell reproduction can be reduced.

Several different HER family receptor inhibitors are in clinical use.

The monoclonal antibodies cetuximab, trastuzumab and pertussumab are directed against the extracellular domain of the target receptor, while lapatinib, gefitinib and erlotinib interfere with the kinase activity of the target proteins *. Other possible treatments for the HER2 subtype include taxane-based chemotherapy (unless the patient has serious heart problems), tyrosine kinase inhibitors, antibody-chemotherapy conjugates, heat shock protein inhibitors, and antibodies that prevent the formation of HER2-HER3 dimers.

Trastuzumab (Herceptin™) is a monoclonal antibody used alone or in combination with cytotoxic chemotherapy in patients with early HER2 overexpressing breast cancer *. In addition, Herceptin™ helps the immune system to better recognize cancer cells and thus help to destroy them.

Pertuzumab is another monoclonal antibody. While trastuzumab prevents HER2 ligand-independent signaling, pertuzumab interferes with HER3 ligand-dependent signaling. Combined anti-HER2 blockade with trastuzumab and pertuzumab in preclinical models shows a synergistic effect *, and in patients with trastuzumab-resistant metastatic HER2-positive breast cancer, shows a clinically positive effect in 50% of cases *.

Although HER2-blocking drugs can inhibit the development of HER2-positive tumors, in most patients, tumors become resistant to targeted therapy within 1 year, leading to a further burst of tumor growth. Resistance to HER2 drugs can result from impaired activation of signaling pathways * such as mutations in the PI3K gene or loss of PTEN phosphatase function * *, resulting in cells not responding to trastuzumab and lapatinib *. In preclinical models, the addition of PI3K and/or mTOR inhibitors restores sensitivity to anti-HER2 drugs *. In patients with HER2-overexpressing metastatic breast cancer previously treated with trastuzumab, the combination of the mTOR inhibitor everolimus with paclitaxel and trastuzumab provides antitumor activity with an overall response rate of 44% *.

Another pathway for developing resistance to trastuzumab is through overexpression of the β2-adrenergic receptor (β2-AR). Co-treatment with trastuzumab and a β-blocker (propranolone) significantly improves progression-free survival as well as overall survival in patients with HER2 metastatic breast cancer *. Other beta-blockers may also be used and may significantly increase progression-free survival *. Because beta-blockers lower blood pressure, they may be most beneficial for patients with hypertension.

A potential mechanism for resistance to HER2 monoclonal antibodies is also the formation of altered forms of the HER2 receptor that lack the binding domain of trastuzumab; such mutated tumors may be resistant to monoclonal antibody therapy but retain their sensitivity to kinase inhibitors *.

Abemaciclib (Verzenio™), an inhibitor of cyclin-dependent kinases Cdk4 and Cdk6, is able to restore the sensitivity of cancer cells to HER2-blocking drugs. Studies in mice have shown that the combination of abemaciclib and HER blocking drugs (trastuzumab) is several times more effective in stopping the growth of HER2-positive tumors than either drug alone *.

• Similarly, lapatinib, a tyrosine kinase inhibitor of HER1 and HER2, may be useful in patients whose disease progresses on trastuzumab *.

Lapatinib in combination with capecitabine has been shown in phase III clinical trials to increase progression-free survival in patients with advanced HER2-positive breast cancer who have previously failed anthracyclines, taxanes, and trastuzumab *. However, tyrosine kinase inhibitors have the potential to cause therapeutic resistance over time.

Recent studies have concluded that only combined HER2 and HER3 blockade will be effective in the treatment of HER2+ breast cancer * *. Meanwhile, effective HER3 inhibitors are still in development.

Further, HER2 overexpression induces the activation of the pro-inflammatory factor NF-κB *, which in turn can upregulate HER2 gene expression, causing radio- or chemo-resistance in HER2+ cancer cells *. Thus, the combination of NF-κB inhibitors and trastuzumab, breaking this mutual support, may be a promising strategy for the treatment of HER2+ breast tumors. Anti-inflammatory therapy, apparently, can also significantly affect the success of therapy for HER2 tumors.

Mathematical modeling predicts that IL-6 and HER2 inhibition is the most effective combination to eliminate breast CSC populations and may lead to treatment success in HER2+ breast cancer *.
Boswellic acid, metformin, rosuvastatin, garlic, cayenne pepper, paprika, and ginger can all contribute modestly to lowering IL-6 levels.

Chaperone (heat shock protein) inhibitors, in particular the HSP90 chaperone, cause proteasomal degradation of the HER2 protein *. Phase II clinical trials have demonstrated the activity of the HSP90 inhibitor tanespimycin in combination with trastuzumab in patients with breast cancer that progressed on trastuzumab monotherapy *.

Nelfinavir, by inhibiting HSP90, selectively inhibited in vitro growth of HER2+ breast cancer cells, including trastuzumab- and/or lapatinib-resistant cell subpopulations, at concentrations used to treat HIV-infected patients *.

• The triptolide contained in thunder duke vine ( Tripterygium wilfordii) inhibits HSP70 and HSP90, significantly reducing the migration and invasion of cancer cells in patients (1 mg/kg *).

• Ethanol extract of LicoriceGlycyrrhiza glabra) downregulates HSP90 gene expression, inducing apoptosis of colon cancer cells in vitro (200 μg/ml) *.

HER2 receptor modulators. Several natural HER2 receptor modulators are known. Used alone, they are certainly less effective than monoclonal antibodies. However, they can enhance the action of therapeutic receptor modulators. Almost all of them are food components, which allows to suppress HER2 signaling to a certain extent due to changes in food preferences.

Black rice extract, due to its content of cyanidins (peonidin-3-glucoside, and especially cyanidin-3-glucoside), which inhibits the proliferation of HER2 cells in vivo more than other studied natural substances *. Black rice hull dry extract containing 25% cyanidins is inexpensive and available online. Dosage: 150-200 mg/day *.

Sulforaphane in the form of broccoli sprouts (4% of the food volume) inhibits the growth of the grafted tumor in mice by 42%, including due to the suppression of HER2 *. Dosage: 200 g/day of broccoli sprouts *.

Gamma-linoleic acid, a polyunsaturated fatty acid ω-6, contained in the seed oil of Evening primrose (Oenothera), Borage (Borago officinalis) and oilseed Hemp (Cannabis sativa) in vitro inhibits the transcription of the HER2 oncogene * * *, and synergistically increases the sensitivity of HER+ cells to trastuzumab up to 30-40 times *. Dosage: 1.8-3 g/day.

Oleic acid, a ω-9 monounsaturated fatty acid predominant in olive and canola oils, in vitro causes a significant decrease in the expression of the HER2 oncogene *. Rats on the olive oil diet showed lower levels of HER2 mRNA expression compared to rats on the corn oil diet *. The combination of oleic acid with monoclonal antibodies (trastuzumab) synergistically enhances the in vitro effect of the latter *. In addition, it was found that oleic acid in vitro is able to inhibit the activity of telomerase, an enzyme that contributes to the «immortality» of cancer cells *.

Polyphenols in extra virgin olive oil, mainly oleuropein-aglycone, prevent in vitro malignant transformation of breast epithelial cells caused by HER2 *, increase the effectiveness of trastuzumab by up to 50 times and allow almost complete restoration of sensitivity to trastuzumab of HER2+ cells *. Based on this, it can be assumed that unfiltered olive oil can provide more benefits than filtered.

EGCG in vitro inhibits the growth of HER2 breast tumor cells * and overcomes HER2 resistance to trastuzumab *. EGCG strongly inhibits in vitro the invasive properties of HER2+ breast cancer cells, promoting a more epithelial phenotype *. EGCG dosage: 500 mg/day (equivalent to 6 cups of green tea per day) *.
Black tea polyphenols in vivo attenuate the resistance of breast cancer HER2 cells to tamoxifen *. In addition, they also inhibit the proliferation induced in MCF-7 cells by dehydroepiandrosterone (DHEA).

Resveratrol and ellagic acid in vitro (10 μM) are able to reduce the activation of HER2 and HER3 receptors. Pre-treatment with resveratrol or ellagic acid, starting 48 hours before treatment with cisplatin, leads to a significant (2-3-fold) increase in cell sensitivity to primary cisplatin therapy. And the constant presence of resveratrol or ellagic acid for 26 weeks of treatment of ovarian cancer cells with cisplatin (A2780) in vitro completely prevents the development of resistance to cisplatin *.

Curcumin (50 μM) triggers apoptosis and blocks cell migration in MDA-MB-231/HER2 cells. Mice treated with curcumin (1 mg/kg 3 times a week) for 21 days had markedly reduced growth of the grafted tumor *.

Piperine inhibits the expression of the HER2 gene at the transcriptional level, due to which it strongly inhibits proliferation and induces apoptosis of HER2+ cells of a cancerous tumor *.

Other means.

Metformin inhibits the activation of the HER2/HER3/Akt signaling pathway as well as cell proliferation and colony formation *. In terms of the effectiveness of prolonging the survival of patients with HER2+ tumors, metformin is able to compete with hormonal or chemotherapy * *.

Aspirin increases the effectiveness of metformin in the treatment of HER2+ breast tumors *.

Melatonin, called the sleep hormone (20 mg/day orally at night), significantly increases the partial response rate to tamoxifen treatment and 1-year survival rates in patients with ER tumors *.

Amygdalin, according to some in vitro studies, may be a promising candidate as a complementary treatment for breast cancer, especially in HER2+ cells*.

Rosmarinic acid (carnosic acid) strongly inhibits in vitro proliferation of ER human breast cancer cells and induces cell cycle arrest in the G1 phase *.

Actein, a triterpene glycoside from Black cohosh (Actaea racemosa), is selectively active against breast cancer cells * and, at low concentrations, synergistically enhances the effects of various chemotherapeutic agents such as doxorubicin, 5-fluorouracil * and digitoxin *. Actein (5 μg/ml) * induces apoptosis in vitro in HER2-overexpressing breast cancer cells (MDA-MB-453 and MCF-7) *.

Black cohosh extract (standardized to 27% triterpene glycosides) can serve as a prophylactic. Female rats given it orally (7-35 mg/kg) showed a strong dose-dependent reduction in the incidence of spontaneous mammary adenocarcinoma *. In human equivalent, this dosage will be 300 mg/day of the extract.

In addition, oral administration of 10 mg/kg of actein for 7 days significantly reduced angiogenesis in experimental mice, and administration of 15 mg/kg for 28 days led to a decrease in the growth of the mouse mammary tumor (4T1) by 36.4%; and also suppressed its metastasis to the lungs and liver – by 57.5% and 43.4%, respectively *. In human terms, this is 60 mg/day of actein.

Hesperidin  *, naringenin  *, and geldanamycin  * inhibit HER2 tyrosine kinase activity in vitro.

Diet, as already mentioned, plays an important role in the containment of tumors. Animal studies show that in HER2 tumors, ω-6 fatty acids (safflower oil) usually have the most adverse effect compared to all others (even worse than saturated fatty acids), while fish oil and olive oil, on the contrary, are the most beneficial *. Observational studies support this association for all other breast cancer subtypes, but also highlight the importance of the ω-3:ω-6 ratio of PUFA *.

Combined therapy.

AR (androgen receptor). Simultaneous inhibition of other receptors may enhance the therapeutic effect of HER2 therapy. The combination of the AR antagonist flutamide and the HER2 inhibitor lapatinib enhances the suppression of HER2+ cell proliferation *. DHEA inhibited the growth of ER, PR, AR+ breast cancer cells in vitro, causing their apoptosis *, but no clinical studies have been reported.

PTP1B (protein tyrosine phosphatase 1B) is a non-receptor tyrosine kinase that is overexpressed in breast cancer cells in most cases, especially in HER2 tumors *.
Ertiprotafib, which is used to treat type II diabetes, is an effective inhibitor of PTP1B. It has also been reported that natural substances such as oleuropein *, curcumin (100 μM) *, alpha-lipoic acid (500 μM, negligible) *, Licorice root (Glycyrrhiza glabra) and others can also exhibit an inhibitory effect in vitro *.

STAT3 (signal transducer and transcription activator 3). Overexpression of HER2 leads to STAT3 phosphorylation, which in turn leads to increased stem cell markers Oct-4, Sox-2, and CD44, tumorigenesis, and resistance to trastuzumab. Inhibition of STAT3 suppresses the CSC phenotype and results in reduced tumor formation and reduced stem cell markers. Combined inhibition of HER2 and STAT3 has shown a synergistic effect on cell growth inhibition in vitro *.
In addition to specific STAT3 inhibitors, some natural substances such as metformin *, quercetin *, narinigenin *, kaempferol *, resveratrol *, apigenin *, curcumin, epigallocatechin gallate and their combinations * are capable of suppressing STAT3 expression.

GPER (G-protein coupled estrogen receptor). Expression of GPER in HER2 tumors is upregulated, stimulating EGFR, which is associated with increased survival, growth, and invasion of tumor cells into surrounding and/or distant tissues. GPER+ tumors showed higher levels of HER2 expression than GPER tumors *.

CB2R (CB2 cannabinoid receptor) is significantly more strongly expressed in the HER2+ subtype than in other breast cancer subtypes. An association has been found between increased CB2 expression in HER2+ tumors and poor patient prognosis. HER2 upregulates CB2 expression by activating the transcription factor ELK1, and overexpression of CB2 upregulates HER2 oncogenic signaling at the level of c-SRC tyrosine kinase *. Gene knockout of CB2 worsens tumor development in mice. There are currently no legal cannabinoid receptor modulators available.

OGF (opioid growth factor) delays the cell cycle in the G1/S phase by inhibiting cyclin-dependent kinase.
Naltrexone is an opioid receptor antagonist (OGFR) used to treat alcohol and drug addiction, as well as to treat multiple sclerosis and autoimmune diseases. While high doses of naltrexone cause complete blockade of opiate receptors, low doses of naltrexone have the opposite effect, a short-term blockade of opiate receptors, which further stimulates an increase in endogenous opiate production.

Low doses of naltrexone delay tumor growth by increasing the amount of OGF and the density of opiate receptors OGFR on the membranes of tumor cells, which makes them more sensitive to endorphins, as well as by increasing the level of endorphins (methionine-enkephalin and beta-endorphin) in the bloodstream. Endorphins, in turn, increase the number and activity of immune cells – natural killer (NK) * and activated T-lymphocytes *.

Naltrexone can also inhibit angiogenesis * and inflammation *, but should not be used in hypothyroidism or in conjunction with opiates. In addition, cells pre-treated with naltrexone for 48 hours were more sensitive to the cytotoxic effects of a number of common chemotherapeutic agents. For example, pretreatment with naltrexone prior to oxaliplatin therapy tripled cancer cell death in vitro *. Vitamin D *, alpha-lipoic acid (ALA), and honokiol have been suggested as supplements to low-dose naltrexone. Remission can occur only after 6 months of use, so for fast-growing tumors, naltrexone, apparently, will be less successful. In addition, the positive effect of naltrexone can be significantly reduced by hormone therapy.

Dosage *: Starting at 1.5mg with an addition of 0.5mg per day until reaching 4.5mg; Take once a day in the evening before bed. The usual pharmacy packaging contains 50 mg in each unit, so accurate weighing is required so as not to exceed the maximum dose.

Triple negative breast cancer.

Triple negative cancer (TNBC) is an aggressive subtype of cancer with a poor prognosis, occurring in up to 20% of all breast cancers. TNBC usually grows faster than other tumor subtypes and is therefore less likely to be seen on an annual mammogram, and more likely to spread to other areas of the body until it is found. Despite the best efforts of medicine, the mortality of patients with TNBC is still twice as high as that of patients with ERα-positive tumors *.

Triple negative cancer is more common in younger women; presumably because primary TNBC tumors present significantly higher levels of BRCA1 hypermethylation compared to healthy tissue or other subtypes of breast tumors *. The fibroblast growth receptor FGFR1 is overexpressed in up to 5.5% of patients with TNBC *. The FGFR2 gene, alleles of which are associated with the risk of postmenopausal breast cancer *, is also overexpressed in 5% of patients with TNBC *.

TNBC exhibits a wide variety of phenotypes. About 70% of TNBCs are basaloid and the remainder are other biologically distinct subtypes * and may have significant differences in such indicators as EGFR, c-KIT, NGFR, CK5/6, CK8/18, vimentin, laminil, p63, nestin, osteonectin, caveolin-1 * *. Based on the analysis of the RNA and DNA profile, they can be divided into six subtypes * *:
- basaloid 1 (BL1), more sensitive to platinum-based chemotherapy and DNA damage (line MDA-MB-468);
- basaloid 2 (BL2), less sensitive to chemotherapy and characterized by increased activity of genes involved in the growth factor signaling pathway;
- mesenchymal (M), sensitive to PI3K signaling pathway inhibitors (BT-549 line);
- mesenchymal stem-like (MSL) (line MDA-MB-231);
- luminal androgen receptor (LAR), more sensitive to androgen receptor antagonists and relatively insensitive to standard chemotherapy (line MDA-MB-453);
- immunomodulatory (IM), characterized by a weakening of the T cell response due to increased expression of CTLA-4 and PD-1 (DU4475 line).
For each of these subtypes, specific potential targets * * can be suggested.

Targets for difference subtypes TNBC Open in new window

Unfortunately, clinics are not yet able to perform a similar analysis of the TNBC tumor subtype in each patient, which makes it impossible to apply specific therapy.

Since receptor-positive tumors develop resistance during treatment and also become refractory to receptor-targeted therapy, TNBC treatment strategies have attracted particular attention. Approaches and techniques that are effective in TNBC may enhance therapy in other subtypes of breast tumors. If they work for TNBC, then they are likely to work for less aggressive cancer subtypes.

However, even when using individual therapy, it should be borne in mind that cancer cells very quickly produce more resistant subclones, evading therapies aimed at them due to bypass metabolic pathways. Thus, by emphasizing the specific sensitivity of the main clone of tumor cells, it will apparently be more effective to simultaneously use, albeit to a lesser extent, other antitumor therapies.


Standard chemotherapy remains the mainstay of treatment for metastatic TNBC, but rapid development of chemotherapy resistance is common in these tumors. TNBC is inherently more chemically resistant than other breast cancer subtypes. Compared to any other cancer subtype, TNBC patients treated with single or combination chemotherapy have lower response rates and shorter remission periods.

The main problem in the treatment of TNBC is the lack of an explicitly unique therapeutic goal for it. TNBC is negative for all three receptors discussed above: ER, PR, and HER2. Thus, the therapeutic options for TNBC are even narrower.

An increase in progression-free survival was observed when chemotherapy was combined with bevacuzimab and iniparib *. Phase III clinical trials involving 236 patients have shown that the combination of cisplatin and gemcitabine is an alternative or even preferred first-line strategy for patients with metastatic triple-negative breast cancer *.

Among other drugs, the greatest efficiency was observed in the appointment of anthracyclines and taxanes *. This seems natural since most TNBCs are two basaloid subtypes, one enriched for cell cycle checkpoints and DNA damage, and the other enriched for genes involved in the growth factor pathway *. Inhibitors of PARP1 (a molecule involved in the repair of damaged DNA), such as olaparib and iniparib, can also increase the overall survival of patients * *, including those with mutant BRBC1 *. Phase III clinical trials (metastatic cases of HER2 BRCA-mutated breast cancer) documented an increase in median progression-free survival and a reduction in the risk of cancer progression and death with olaparib therapy compared with standard chemotherapy *.

Bevacizumab is a humanized monoclonal antibody directed against all VEGF-A isoforms.

Salinomycin is a selective inhibitor of CSC, acting by inhibiting the Wnt signaling pathway and degrading LRP6 *, but it has not reached clinical trials.

Clofazimine is a well-known antibiotic used in the treatment of tuberculosis. It also inhibits Wnt signaling, and although it does not kill cancer cells, it greatly inhibits their growth in animal studies *. The equivalent human dosage used in this study is 1'200 mg/day.

Doxycycline is an antibiotic with a successful long history of use. It weakens cancer cells by inhibiting mitochondrial respiration. The combination of doxycycline with high intravenous doses of vitamin C, which inhibits glycolytic respiration, is lethal to CSCs *.

Targeted therapy.

Metabolic pathways of the cell Enlarge Image

As already noted, TNBC is not a single disease, but rather a combined group of diverse subtypes, united only by the absence of ER, PR and HER2 expression, but showing great diversity in other markers *. This causes the difficulty of choosing TNBC therapy, which, in the absence of appropriate analyzes, may be exacerbated by the lack of understanding which of these markers can be emphasized in each case.

The absence of the main therapeutic targets (ER, PR and HER2) forces one to resort to additional strategies *, however, they are often ineffective. Although the distinctive features of certain types of TNBC cells may be weak, it is worth using each of them. Moreover, combinations of several low effective therapies, acting cumulatively or synergistically, can have a significant positive effect.

A whole network of molecular targets for TNBC * has been explored, including several receptors, poly(ADP-ribose) polymerase (PARP), vascular endothelial growth factor (VEGF), protein tyrosine kinase, phosphatases, proteases, PI3K/Akt signaling pathway, microRNA (miR) and long non-coding RNAs (lncRNAs). As well as some targets used in HER2 therapy, such as ALDH1.

Receptor tyrosine kinases (RTKs) that may be potential targets in TNBC include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and MET.

EGFR (epidermal growth factor receptor, HER1). More than 60% of basaloid tumors * *, and up to 50% of TNBCs * are characterized by EGFR expression. Tyrosine kinase inhibitors (TKi) such as gefitinib and erlotinib inhibit in vitro and in vivo the proliferation of EGFR-overexpressing cancer cell lines, including TNBC (MDA-MB-231) *, by more than 50%*.

Oral administration of gefitinib (Iressa™) inhibited the growth of grafted TNBC (MDA-MB-231) and HER2 (SKOV3) tumors in mice by 71% and 32%, respectively, without increasing cancer cell apoptosis *.

Phase II clinical trials in patients with advanced cancer showed that gefitinib (500 mg/day) stabilized the disease within 6 months in 38.7% of patients by inhibiting EGFR phosphorylation *. Breast tumors that developed tamoxifen insensitivity stopped growing within 6 months in 54% of patients treated with gefitinib. However, only 11.5% of patients with ER-negative tumors showed disease stabilization when treated with gefitinib *.
Anti-EGFR therapy in addition to carboplatin using the cetuximab (monoclonal body) has also shown insufficient efficacy in clinical trials *.

The addition of chemotherapy with a PARP inhibitor increased overall survival in patients with metastatic TNBC compared with chemotherapy alone in phase II clinical trials *, but these results were still not good enough.
mTOR inhibition has proven to be a more effective aid to EGFR inhibitors. The combination of the EGFR inhibitor lapatinib and the mTOR protein kinase inhibitor rapamycin synergistically provides an increase in cytotoxicity than either alone in vitro and significantly reduces the growth of TNBC in vivo. The combination of rapamycin (3 mg/kg, twice a week) and lapatinib (75 mg/kg, orally, 5 days a week) approximately halved the growth of TNBC tumors inoculated into mice (MDA-MB-231 and MDA-MB-468) *.

Several flavonoids (cyanidin, delphinidin, quercetin, ellagic acid, EGCG, pterostilbene, procyanidins, and resveratrol) reduce in vitro tyrosine kinase activity or prevent EGFR dimerization, and decrease the phosphorylation of certain kinases such as PI3K/Akt and ERK/MAPK. However, there is no certainty that they will be any effective in living humans because EGFR inhibition has not yet proven to be a suitable target for monotherapy in TNBC.
The combination of TKi with monoclonal antibodies is currently being studied, which acts synergistically to reduce cancer cell proliferation (MDA-MB-468 and SUM-1315) *.

Unfortunately, the problem with anti-EGFR therapy is the same as with other receptor therapies. It lies in the fact that many types of normal cells also contain certain receptors. Thus, targeted therapy against them is, strictly speaking, not that targeted. It will inevitably affect cells that are guilty only of being too similar to cancer.

HGFR (hepatocyte growth factor receptor) controls cell invasiveness and plays an important role in the survival of breast cancer patients.
The following natural substances have been reported to inhibit in vitro HGF-induced invasion and metastasis of various cancer cells: apigenin *, EGCG  * *, curcumin  *, madecassoside from (Centella asitica) *, resveratrol *, diosgenin *. Including herbacetin from Ephedra (Ephedrae sinica), against breast cancer cells (MDA-MB-231) *.

FGFR (fibroblast growth factor receptor) and HGFR have been investigated as therapeutic targets for TNBC but have not shown significant success. In this case, optimistic preclinical results were obtained by combining HGFR inhibitors with EGFR or PARP inhibitors *.

VEGFR (vascular endothelial growth factor receptor). Another direction of additional therapy may be the use of anti-angiogenic mechanisms * *.
The anti-VEGF-A monoclonal antibody, bevacizumab, markedly improves response rates and patient survival *. However, strong anti-angiogenic therapy may cause malignant growth over time due to increased invasiveness *. In addition, the use of bevacizumab has been associated with an increase in serious side effects such as stroke, wound healing complications, and organ damage *. Based on these facts, bevacuzimab is not recommended for use in TNBC.

Sorafenib, a multikinase inhibitor targeting VEGFR1, VEGFR2, VEGFR3, PDGFRB, RAF, KIT, and FLT-3 *, in combination with capecitabine showed a marked increase in survival compared to capecitabine alone in a clinical trial *.

Overall, anti-VEGF monotherapy shows very modest results. In addition, the activation of additional pro-angiogenic mechanisms that bypass the effects of therapeutic agents may, over time, lead to the failure of anti-antiogenic therapy *.

c-KIT (CD117, stem cell growth factor receptor) is a receptor thyrokinase that can be expressed in 25-45% of TNBC cases * *. Growth factor receptors VEGF, PDGF and c-KIT are downregulated by the tyrosine kinase inhibitor sunitinib. In combination with paclitaxel, sunitinib produced therapeutic responses in 33% of patients with TNBC *, and in 15% of patients with metastatic TNBC previously treated with anthracycline and taxane *. Other studies have shown more modest results *. In clinical trials, sunitinib showed no measurable effect against c-Kit in TNBC *. The c-KIT receptor can also be blocked in vitro by imatinib * or dasatinib *, but clinical evidence for this has not yet been provided and their possible efficacy is unclear.

Non-receptor tyrosine kinases (NRTKs) that are potential targets in TNBC include the PI3K/Akt/mTOR, Src, and MEK signaling cascades.

Src is a tyrosine kinase, a non-receptor signaling kinase that interacts with several signaling pathways (PDGFR, EGFR, IGF-1R and HGFR) that regulate proliferation, angiogenesis, invasion and metastasis *. It is also deregulated in breast cancer * *. The content of Src in the cytoplasm is higher in TNBC cells than in ER+ cells *.
Unfortunately, clinical trials of Src inhibitors on their own, such as dasatinib, saracatinib and bosutinib, have not shown clinical benefit in TNBC * * *. However, adding them to other chemotherapeutic substances enhanced the effect of the latter. Thus, the addition of dasatinib to cetuximab and cisplatin enhanced the inhibition of cell growth, migration, and invasion *.

PI3K/Akt/mTOR is a signaling pathway that regulates cell proliferation and survival *. Activation of PIK3CA mutations is observed in approximately 40% of AR+ TNBC tumors compared to 4% of AR TNBC tumors *. Inhibition of mTOR may increase the sensitivity of resistant HER2+ tumors to trastuzumab *. Although mTOR inhibition with rapamycin or everolimus in vitro increased the cytotoxicity of various chemotherapeutic agents in several TNBC lines * *, phase II clinical trials of the combination of everolimus and chemotherapy (5-fluorouracil, epirubicin, cyclophosphamide, and paclitaxel) in a primary TNBC tumor showed no measurable therapeutic effect *. However, the combination of PI3K inhibition can be used to enhance the antitumor activity of other agents such as PARP inhibitors *.

Metformin suppresses mTOR signaling in several cancer cell lines in vitro *. Metformin moderately inhibits the growth of grafted TNBC tumors in mice *. The combination of metformin with doxorubicin acts synergistically. After 15 days of treatment (100 mg/l metformin in drinking water), this combination virtually eliminated grafted tumors in mice. While at the same doses, doxorubicin alone causes a 2-fold decrease in tumor volume, and metformin alone has an insignificant effect *. The equivalent human dose will be 100 mg/day.
The combination of signaling silencing metformin with inflammation suppressing aspirin produces a synergistic effect in vitro in TNBC cells to reduce cell viability and colony formation *.

Simvastatin and other statins widely used to lower cholesterol levels suppress the PI3K/mTOR signaling pathway in breast cancer in vitro. They deactivate mTOR in both ER+ tumors and TNBC; however, TNBC cells appear to be more sensitive to statin treatment than ER+ cells *. The combination of simvastatin with pentoxifylline synergistically enhances cancer cell death *.

Wortmannin *, silibinin *, fisetin * and resveratrol * have also been shown to be inhibitors of PI3K in vitro, but this has not yet been clinically proven.

One study identified kinases that are abnormally expressed and critical for the growth of ER-negative breast cancer *. In particular, a group of kinases involved in cell cycle checkpoint control and mitogenesis (Chk1, bub1, TTK and Ak2); as well as a group of kinases involved in the S6 kinase signaling pathway (RPS6KA3, SMG-1 and RPS6KA1). Of the 20 kinases examined, 14 were critical for the growth of ER-breast cancer cell lines and could potentially be used therapeutically. Turning off 9 of them (EPHB4, LIMK2, DAPK1, YES1, RYK, VRK2, PTK7, RAF1, UCK2) has a significant inhibitory effect on ER cells (MDA-MB-468 and MDA-MB-231), but does not affect ER+ breast cancer cells. Blocking another 5 kinases (bub1, Chk1, IRAk1, CCL4, TTK) inhibits the growth of all breast cancer cell lines. At the same time, deactivation of MPZL1 kinase has a strong proliferating effect on all breast cancer cell lines.
The in vitro kinase inhibitor dasatinib blocks the activity of many of the kinases identified in this assay, including YES1, EPHB4, and EPHA2.

MAPK (mitogen-activated protein kinase). The MAPK signaling pathway is also being explored as a therapeutic target in TNBC. In vitro, MEK kinase inhibitors significantly reduced TNBC * invasion and inhibited lung metastasis * in mice. One clinical study reported a complete response in a TNBC patient treated with the MEK inhibitor trametinib in conjunction with gemcitabine *. Another FDA-approved inhibitor is cobymethinib. Unfortunately, anti-PI3K and anti-MEK therapies develop drug resistance. However, their combination enhances each other's action against breast cancer basaloid cells * * and reduces their toxicity.

CDK (cyclin-dependent kinases). Some observations show that in certain situations, restriction of CDK activity suppresses the proliferation of cancer cells, but not the division of normal cells *. CD44+/CD24 inflammatory breast cancer stem cells that were resistant to conventional chemotherapy showed in vitro sensitivity to a specific inhibitor of cyclin-dependent kinase CDK2 *. Thus, CDK inhibitors could enhance the effectiveness of conventional chemotherapy. CDK inhibitors usually show high toxicity, but recently several CDK inhibitors with more benign side effects have entered clinical trials. These are, for example, palbociclib and dinaciclib.

Several natural CDK inhibitors have been identified, and some of them are quite selective and effective. They inhibit cell division by delaying the cell cycle in the G1 and/or G2/M phases. And besides, they are able to promote apoptosis in proliferating cells.

Gamma-butyrolactone (γ-butyrolactone), a CDK inhibitor, is found in small amounts in red wine and mung beans. For some time distributed as a supplement for athletes, but in 1990 it was banned by the FDA as a psychotropic substance.

Indirubin from Indigo (Indigofera tinctoria) and from Woad due ( Isatis tinctoria) plants also acts as an inhibitor of CDK in vitro *. However, indirubin exhibits poor solubility, low absorption, and gastrointestinal toxicity. No clinical trials of indirubin have been reported.

Flavopiridol, a flavonoid alkaloid found in the plants Harinhara ( Amoora rohituka) and Lassuni amari (Dysoxylum binacteriferum), also has some other antitumor effects. The recommended doses of flavopiridol as a 1-hour infusion derived from phase I clinical trials were: 37.5 mg/m2 for 5 days, 50 mg/m2 for 3 days, and 62.5 mg/m2 for 1 day *. In a subsequent clinical study, the combination of docetaxel and flavopiridol (26 mg/m2) as a 1-hour infusion daily for 3 days produced a partial response in some patients with metastatic breast cancer, but flavopiridol exhibited severe clinical toxicity *.

PARP. Poly(ADP-ribose) polymerase plays an essential role in DNA damage recognition and repair. Inhibitors of this enzyme are used to treat TNBC patients with BRCA1 or BRCA2 mutations, but may be effective in other types of TNBC *.

Combinations of PARP inhibitors with chemotherapy increase the therapeutic response.

Iniparib, a PARP-1 inhibitor, has been shown to significantly increase the efficacy of carboplatin and gemcitabine * * in clinical trials, but its success is still questionable *.

Olaparib, another PARP-1 inhibitor, has been more successful. In clinical studies, it has provided objective responses in women with BRCA-deficient breast cancer *. In advanced breast cancer (more than half of which was TNBC), in patients with BRCA1 and/or BRCA2 deficiency treated with olaparib (400 mg orally twice daily), the overall response rate was 41% *.

To date, PARP inhibitors seem to be the most promising direction in addition to other TNBC therapies aimed at irreversible damage to the DNA molecule.

Non-classical receptors. TNBCs lack estrogen receptor α (ERα), progesterone receptor (PR), and do not overexpress human epidermal growth factor receptor type 2 (HER2). They are not responsive to hormone therapy or targeted therapy using anti-HER2 antibodies (such as trastuzumab). However, they may express other, non-classical receptors that may be subject to therapeutic intervention.

GPER (G-protein coupled estrogen receptor) is very commonly expressed in TNBC and is an indicator of poor prognosis *. GPER is common in TNBC associated with young age and possible malignant recurrence *. Up to 70% of IBC patients express GPER, while ER, PR, HER2, and EGFR expression was found in 43%, 35%, 39%, and 34% of patients, respectively *. However, the causal relationship between their expression and disease is not clear. There is no unambiguous evidence that a high level of GPER is a stimulator of breast tumors, and not vice versa, has a protective role.

GPER expression in vivo decreases as a breast tumor develops, which may indicate its antitumor role *. Low expression of the GPER protein is significantly associated with the aggressiveness of tumor behavior and with poor survival of breast cancer patients *. An increase in expression with some agonists leads to an increase in proliferation, while others lead to its decrease. Due to the specific role of the GPER, there are no clear recommendations as to the direction of its regulation.
There are currently no clinically tested selective modulators of GPER-1 expression. It has been noted that GPER expression parallels EGFR expression, and EGFR inhibition with gefitinib can simultaneously dramatically reduce GPER expression in vitro (up to 85% in HCC70 cells) *. However, in clinical trials, gefitinib was found to be ineffective in ER tumors *.

There are several naturally occurring GPER modulators.

Baicalein has an inhibitory effect on both ERα and GPER estrogen signaling, apparently by blocking estrogen binding to the receptor through competition. In in vitro studies, baicalein (10 μM) inhibits estradiol-induced migration, adhesion, and invasion of ER+ (MCF7) and ER (SK-BR-3) breast cancer cells *. In addition, baicalein abolished the estradiol-induced malignant transformation effects of normal breast cells; that is, it can be an effective preventive agent if it proves its antitumor effect in clinical trials.

Of the 48 known nuclear receptors *, only ER, PR, AR, RAR, ROR and VDR* are known to be associated with breast cancer *.

AR (nuclear androgen receptor) is expressed in up to 90% of ER+ tumors, to a lesser extent in HER2 tumors *, and in about 10-35% of TNBC tumors * * *. AR activity in breast tumors is widespread and higher than ER or PR activity * * *. AR activates the Wnt/β-catenin pathway, which leads to an increase in HER3 and has been implicated in breast tumorigenesis *.

AR expression is considered to be a favorable biomarker for ER+ breast tumors * * *, i.e. AR is a protective protein. Reduced AR expression in ER+ tumors may predict an increased risk of breast cancer recurrence and death *. Conversely, AR agonists are inhibitors of ER-positive breast tumors *.
AR-mediated activation of HER2/HER3 transmission results in upregulation of MYC gene activity, which increases the transcriptional activity of androgen response genes in ER and AR+ cancers *.

At the same time, the role of ARs in TNBC remains uncertain, apparently due to their large disease heterogeneity, as well as the presence or absence of other signaling mechanisms. In the presence of ERα, the AR may antagonize its effects, while in the absence of ERα, the AR may exhibit an ERα-mimic effect, promoting a tumor *. In patients with TNBC, AR expression was negatively associated with GPER expression, and positively associated with tumor size and lymph node metastases. AR expression resulted in tumor progression in some subtypes of TNBC models * as well as in AR+/ER cancer cells *. In ER+/PR+, AR+ tumors, androgen treatment causes growth suppression, while in ER/PR, AR+ tumors, androgens cause the opposite effect * * *.
Growth of some ER+ breast cancer cell lines (MCF-7), like TNBC (MDA-MB-453), is also stimulated by androgens and inhibited by antiandrogens * *.

Antiandrogen agents are used to treat prostate cancer, and can be used to treat TNBC varieties that express AR positively *. Targeted therapy with second-generation synthetic antiandrogens, such as bicalutamide and enzalutamide, inhibited in vitro proliferation, invasion and migration in various TNBC cell lines *.

Bicalutamide is used as an androgen antagonist in prostate cancer. In a phase II clinical trial in patients with metastatic ER/PR/AR+ breast cancer, a 6-month clinical efficacy of bicalutamide (150 mg/day) was 19% *. A study of the effect of paclitaxel, 5-fluorouracil and cyclophosphamide in AR+ TNBC showed that in the presence of androgen, cancer cells have a significantly higher survival rate and less apoptosis, which can be overcome by the addition of bicalutamide *. The combination of bicalutamide with agents targeting the EGFR, PDGFRβ, PI3K/mTOR and ERK pathways creates a synergistic effect *. Combination therapy with PI3K and AR inhibitors has a cumulative apoptotic effect in AR+ TNBC cells *.

Enzalutamide binds the androgen receptor with greater affinity than bicalutamide and exhibits a higher and more varied effect. It significantly reduces proliferation, growth, migration, and invasion in vitro, and increases apoptosis in four TNBC lines (SUM159PT, HCC1806, BT549, and MDA-MB-231) representing the non-LAR subtypes of TNBC (mesenchymal and basaloid). Supplied with feed (50 mg per 1 kg of food), enzalutamide significantly reduces the viability of grafted SUM159PT and HCC1806 tumors in mice *.
Enzalutamide inhibits androgen-induced growth of both ER-positive and ER-negative AR-expressing cancers and provides a positive response in approximately 40% of patients *.

• In the clinical therapy of prostate cancer, in addition to bicalutamide and enzulutamide, abiraterone is also used to regulate sex hormones. Abiraterone is an inhibitor of the enzyme 17α-hydroxylase, which is involved in the formation of androstenedione, the precursor hormones of testosterone and estrone, and further, dihydrotestosterone and estradiol. Presumably, complete inhibition of androgen and estrogen signaling may provide a better effect against breast cancer. However, in a 6-month clinical study, the combination of 1'000 mg abiraterone with 5 mg prednisone showed a 20% therapeutic response in AR+ TNBC patients, which included one complete response and five cases of disease stabilization. And the overall response rate was 6.7%, much lower than enzalutamide *.

Natural AR agonists are piperine (black and red pepper), apigenin, luteolin *, as well as hesperidin (citrus fruits), coumaric acid (tea, red wine), tanshinon (sage) and some other substances, but their effectiveness is 4 orders of magnitude lower than synthetic agonists *.

So, in non-TNBC breast tumors, androgens, and in particular non-steroidal AR agonists, may have a beneficial effect. At the same time, despite all the above positive effects of antiandrogens in most TNBC tumors, due to the diversity of their phenotypes, some subtypes can respond to antagonists, while other subtypes can theoretically respond to agonists. Thus, the benefit of antiandrogens in all cases of TNBC cannot be considered definitively proven.

RAR (nuclear retinoic acid receptor), by binding to its ligand, causes the transcription of genes that regulate cell differentiation *. ATRA treatment of MCF-7 breast cancer stem cells results in cell differentiation, reduced invasion and migration, and increased sensitivity to anticancer treatment *.

ROR (retinoic acid-related orphan nuclear receptors) include three varieties – RORα, RORβ and RORγ. RORα has been identified as a therapeutic target in breast cancer *. Prospective studies have shown that RORα inactivation is associated with poor clinical outcome. Conversely, restoration of RORα expression in breast cancer cells suppresses their malignant and invasive phenotypes in vitro and in vivo *.

Natural RORα ligands are melatonin, cholesterol and cholesterol sulfate *, while RORβ ligands are retinoids such as ATRA *. Expressed by immune cells, RORγ acts as an immune suppressor, and expressed by cancer cells, it acts as a potential survival factor. Vitamin D3 metabolites are inverse agonists for RORα/γ; therefore, RORα and RORγ expression levels can be regulated by both vitamin D3 and ultraviolet irradiation *.

VDR (nuclear vitamin D receptor) may also be considered as a therapeutic target, as approximately 90% of all breast tumors * express VDRs, and approximately two-thirds of TNBC *. Clinical trials of D3 (calcitriol) as a monotherapy for breast tumors have led to the conclusion that a therapeutic dose that provides an antitumor effect is difficult to achieve due to hypercalcemia, kidney stone formation * and vascular calcification *. However, VDR agonists may act synergistically with agonists of other receptors such as AR *. The combination of VDR agonists with taxol inhibits cancer cell proliferation more effectively than taxol alone *.

β-AR (β-adrenergic receptor). β-AR antagonists, by reducing chronic psychological stress, can significantly improve the survival of patients with breast cancer, including TNBC *.

Propranolol, a non-selective β-antagonist widely used in high blood pressure, inhibits in vitro migration of TNBC cells (MDA-MB-231) *, and in experiments on mice with grafted TNBC tumors, reduces metastasis *. An observational study found that in postmenopausal TNBC patients, consumption of beta-blockers was associated with a significant reduction in the risk of recurrence, metastasis, and death associated with breast cancer *.
A manipulative study showed that in patients with hypertension treated with beta-blockers (mainly atenolol), in comparison with other antihypertensive drugs, there was a significant decrease in the development of metastases (by 2.5 times), tumor recurrence (by 2 times) and more. long relapse-free period *.

AHR (aryl hydrocarbon receptor) is overexpressed and activated in many breast cancers * *.

Raloxifene, used to prevent osteoporosis in postmenopausal women, activates AHR and induces in vitro apoptosis of TNBC cells (MDA-MB-231) without affecting normal breast epithelial cells *.

Sulindac, a non-steroidal anti-inflammatory drug, is an AHR agonist and inhibits TNBC (MDA-MB-231) cell invasion and metastasis in vitro *. Dosage: 500 mg/day *.

Tranilast, an anti-inflammatory agent, is an AHR agonist with an inhibitory effect on mammary CSCs. Mice treated with tranilast (300 mg/kg) from days 1 to 21 after tumor implantation showed three times less tumor growth compared to controls and virtually no metastases compared to controls *. The human equivalent dosage that provides the required plasma concentration of tranilast is about 2'000 mg/day.

Omeprazole, used for peptic ulcer disease, is one of the most effective AHR agonists. It reduces invasion and metastasis of TNBC cells (MDA-MB-231) * and strongly inhibits the migration of cancer cells (BT474, MDA-MD-231, MDA-MB-468) in vitro *. Another proton pump inhibitor, lansoprazole, halved the growth of the grafted MDA-MB-231 tumor in mice *.

Diindolylmethane (DIM) is considered the most potent natural AHR agonist, but in clinical studies, daily consumption of cruciferous vegetables (150 g broccoli plus 300 g Brussels sprouts) increased AHR activity by no more than 12% *.

CB1R, CB2R (cannabinoid receptor). Both cannabinoid receptor agonists significantly inhibit the proliferation and migration of MDA-MB-231 and MDA-MB-468 cells in tumor-grafted mice *.

ACEA (selective CB1 receptor agonist) at 200 nM reduces the in vitro invasive activity of CSC TNBC (MDA-MB-231) without affecting proliferative activity *.
Intratumoral injections of cannabidiol (10 mg/kg) for 3 weeks in mice inoculated with triple-negative breast cancer tumors inhibited primary tumor growth by 20% and reduced lung metastasis by a third *.
Combination therapy targets two proteins activated in TNBC: the CB2 cannabinoid receptor (G-protein coupled receptor) and the translocator protein (TSPO, mitochondrial membrane receptor). The combination of a CB2R agonist and photodynamic therapy with TSPO-PDT in vitro resulted in synergistic inhibition in TNBC cells and tumor *.

PD-1 (programmed cell death receptor 1). Tumor antigens are recognized by T lymphocytes, which trigger the immune system's response against cancer cells using molecules called «checkpoints» such as the cytotoxic protein CTLA-4 and the PD-1 receptor. Checkpoints prevent immune cells from damaging normal tissues in the body, but cancer cells take advantage of this to present themselves as normal cells. They upregulate the expression of checkpoint molecules and thus avoid attack by the immune system.

High levels of PD-L1 in tumors lead to immune response evasion and cancer progression *. Conversely, the combination of immunostimulants with CTLA-4 and PD-1 inhibitors renders HER2+ tumors susceptible to immune attack * and appears to be similarly beneficial in TNBC tumors.
Monoclonal bodies that bind to either the cell surface receptor PD-1 on a T cell (nivolumab) or its ligand, PD-L1 on a cancer cell (avelumab), can enhance T cell immune responses to tumor cells * *. Phase I clinical trials of monoclonal antibodies to PD1 (pembrolizumab) and to PD-L1 (atezolizumab) in TNBC patients gave objective therapeutic responses (18.5 and 33%, respectively) * *.

Unfortunately, a large percentage of patients do not respond to such therapy, especially for breast cancer, and besides, it can cause serious autoimmune reactions.

Thymoquinone (3 mg/kg) in mice regulates genes involved in the induction of apoptosis via death receptors *.

Other potential targets for TNBC.

KLF4 (Krueppel-like factor 4, or the zinc-finger transcription factor) plays an important role in regulating various cellular processes including apoptosis, migration and cell stem characteristics * * *. KLF4 supports the migration and invasion of breast CSC cancer cells; and its disabling suppresses tumor development, as well as suppresses the migration and proportion of CSC in the tumor *. More than 70% of breast cancer cases are characterized by increased expression of KLF4, which is associated with an aggressive phenotype in the early stage of breast cancer * *.

Withaferin A in vitro (1-2 μM) inhibits CSC of both ER+ cells (MCF-7) and ER cells (SUM159) of the mammary gland, the latter appearing to be significantly more inhibited. ALDH1 activity and the number of mammospheres were significantly reduced by siRNA KLF4 interference in the SUM159 cell line *.

Intraperitoneal injections of withaferin to mice (5 mg/kg 3 times a week for 28 weeks) did not reduce the overall tumor incidence, however, the palpable tumor size was reduced by half *. Along the way, Withaferin, compared with the control, reduced the ability to form mammospheres of cancer cells, the activity of ALDH1, and the downregulation of such an oncogenic protein as Bmi-1.

Extract Ashwagandha (Withania somnifera) can also be used as a prophylactic, based on the fact that its consumption (150 mg/kg orally for 155 days) reduced the incidence and multiplicity of carcinogen-induced rat ER+ rat's breast cancer *. In addition, the antiestrogenic and proapoptotic effects of withaferin in vitro (2.5 μM) are manifested by a significant suppression of the ERα protein level and a slight increase in ERβ *.

HSP90 (heat shock protein 90) is a chaperone or protective protein that provides the structural maturation of various «client proteins», including EGFR, AKT, PI3K, etc. Thus, inhibition of HSP90 can inhibit multiple signaling cascades that regulate the growth, proliferation and survival of cancer cells. Chaperone inhibitors can be used not only in HER2 positive cancer but also in TNBC. HSP90 chaperone inhibitors such as radicicol *, tanespimycin *, alvespimycin, retaspimycin are under investigation.

Ganetespib is a powerful new generation HSP90 inhibitor that is effective on its own or in combination with other chemotherapy drugs. Two cases of regression of metastatic TNBC cancer due to the use of ganestipib have been reported *. Clinical studies of ganetespib (150 mg/m2 twice a week, 3 of 4 weeks for 12 weeks) have also been reported. Of the 15 patients with TNBC tumor, two patients achieved an overall response, three had stable disease, five had progressive disease, and five were not evaluated. Of the five patients with HER2+ breast tumor, two patients had an overall tumor response, two had stable disease, and one had no score *.

Apigenin, a natural inhibitor of HSP90 chaperones, destabilizes the HIF-1α protein caused by both hypoxia * and metals *, acting independently of oxygen levels *. The chrysin flavonoid also inhibits the interaction of HIF-1α and HSP90 *.

Wogonin in vitro increases HIF-1α interactions with VHL and increases HIF-1 ubiquitation *. However, the effectiveness of any natural inhibitors can hardly be compared with genetespib.

VGSC (voltage-gated sodium channel) is commonly deregulated in various tumors, including TNBC *, which contributes to in vitro invasiveness* and in vivo metastasis *. Several VGSC inhibitors, such as ranolazine (an antiarrhythmic drug), riluzole (an amyotrophic drug), and phenytoin (an antiepileptic drug), inhibit the behavior of metastatic cells in vitro * *. However, it is not yet possible to assess their therapeutic potential against TNBC.

OGF (opioid growth factor) see above.

Naltrexone. Dosage: 4.5 mg once a day in the evening before bedtime.

CTLA-4 (cytotoxic T-lymphocyte antigen 4) is a cell surface receptor for the T-lymphocyte regulator and inhibits T-cell activation. Blocking CTLA-4 with monoclonal antibodies such as ipilimumab and tremelimumab has the potential to increase the activity of T cells against tumor cells *.

Prolactin is a growth factor that stimulates the proliferation of breast cancer cells. Abnormally elevated blood prolactin concentrations are associated with poor prognosis and reduced efficacy of anticancer therapy in metastatic breast cancer.

Bromocriptine is a drug that specifically suppresses the production of prolactin without affecting the level of other hormones produced by the pituitary gland. Oral administration of bromocriptine (2.5 mg/day) daily until the end of Taxotere treatment markedly improved treatment outcomes compared with patients receiving chemotherapy alone *.
Bromocriptine inhibits drug-resistant tumor cells with various resistance mechanisms in vitro in a hormone-independent manner *.

Cabergoline, another prolactin inhibitor, also increased the efficacy of hormone therapy with tamoxifen (1 mg orally twice a week for 4 weeks)*, but it appears to be more modest than bromocriptine *.

LOX (lysyl oxidase) is a group of enzymes that convert polyunsaturated fatty acids into biologically active (pro-inflammatory) metabolites. LOX activity is higher in cancerous than in normal breast tissues * and LOX appears to play a key role in shaping the microenvironment of the precancerous niche *. There is a very strong overexpression of LOX in TNBC compared to other subtypes of breast cancer. In addition, overexpression was observed in less differentiated tumors compared to more differentiated ones *. LOX overexpression was positively correlated with resistance to radiation, doxorubin, and mitoxantrone, but negatively correlated with resistance to bisphosphonate, PARP1 inhibitors, cisplatin, trabectin, and gemcitabine *.

Many herbal remedies are able to inhibit the synthesis or action of LOX * * *, however, they act weakly and non-specifically, affecting other chemical and biological mechanisms. These include thymoquinone from Black cumin (Nigella sativa), aethiopinone from Ethiopian sage (Salvia aethiopsis), cirsiliol from common Sage (Salvia officinalis), gingerol from Ginger, tryptanthrin from Woad dye (Isatis tinctoria), cepaene from Onion (Allium cepa).

NF-κB is a transcription factor whose signaling is often impaired in TNBC *, and thus the NF-κB inhibitors discussed above may reduce inflammatory levels and improve therapeutic efficacy.

Iron is a critical element in the process of cell division. Cancer cells, with their high proliferation rate, require more iron than normal cells, making them more susceptible to iron depletion. For this reason, iron chelators such as deferasirox, deferoxamine or dexrazoxane could enhance the therapeutic effect of chemotherapy drugs such as doxorubicin, cisplatin and carboplatin in triple negative breast cancer * *. Metformin further enhances the effect of iron chelators *. However, there have been no reports of clinical studies in this direction so far.

Epigenetic therapy.

Epigenetic therapy is the dynamic regulation of gene expression without altering the gene sequence. Despite the promise of this therapeutic area, the existing epigenetic modulators are active throughout the genome, and no molecules have been developed that are specifically active at the target DNA site.

Phase II clinical trials of the combination of 5-azacytidine and entinostat in women with TNBC showed no success *. At the same time, the combination of vorinostat with paclitaxel and trastuzumab, followed by doxorubicin with cyclophosphamide, provided a therapeutic response in half of the patients with the HER2+ breast tumor subtype, in a quarter of the patients with TNBC, but in none of the patients with ER+ and HER2 subtypes *.

In a third of TNBC patients, the expression of both the p53 «guardian of the genome» gene and the retinoblastoma tumor suppressor gene (RB1) is simultaneously lost. The gene loss is not medically treated, but the RB1-deficient tumor variant is characterized by increased expression of the mitochondrial protein (MPT) gene, which can be acted upon. For example, salinomycin and the MPT antagonist tigecycline inhibit the proliferation of predominantly TNBC RB1-deficient tumors by targeting not only the cells of the tumor array, but also the stem cell fraction *. However, research on tigecycline has so far been limited to mouse models (dose 75 mg/kg).

A number of food components show their positive epigenetic effect in vitro and in vivo. While carcinogens negatively alter gene expression, promoting malignant transformation and cancer aggressiveness, many natural substances * * can act in the opposite direction, reducing cancer risk *, and their effective dose is sometimes close to that normally taken in the form of food. Among the best studied are EGCG  *, curcumin, genistein * *, resveratrol, EGCG * and selenium  *, DIM  * and sulforaphane  * * * *, curcumin  *, quercetin  *, DADS* and allylmercaptan  *.

Green tea. The combination of green tea polyphenols (0.3% solution of polyphenols in drinking water) and broccoli sprouts (13% of the total weight of the diet) reduces the growth rate of the grafted mammary tumor in mice by 3.3 times due to the epigenetic effect and significantly exacerbates sensitivity to tamoxifen TNBC cells *. The daily dosages of active ingredients used in this study are equivalent to a human consumption of 1-2 cups of green tea (1.5 mg/ml EGCG) * and 133 g (2 cups) of broccoli sprouts *, which is quite achievable without any discomfort.
Postprandial exercise, as well as regular consumption of green tea, was associated with a doubling of survival rates among women with TNBC compared to women who did not *.

• The combination of genistein (DNMT inhibitor) and sulforaphane (HDAC inhibitor) works synergistically to reduce the viability of breast cancer cells (MCF-7 and MDA-MB-231). A dosage equivalent to 2 g/day of genistein and 133 g/day of broccoli sprouts in humans halved the incidence of spontaneous cancer and the volume of breast tumors in mice *.

Withaferin A, found in Ashwagandha (Withania somnifera), causes DNA hypermethylation and H3K4me3 demethylation, inhibiting the aggressive characteristics of TNBC *. Equiv. dosage: 400 μg *.

Pomiferin, contained in Osage orange (Maclura pomifera), is able to inhibit the activity of HDAC *.

Nutritional correction.

TNBC has been reported to be associated with certain micronutrient deficiencies such as zinc, folic acid, and beta-carotene *. Of course, the hope of defeating cancer by taking corrective supplements would be extremely naive, but supplements can also make a modest contribution to the overall positive result of complex therapy.

Multivitamins. Patients with ER tumors who took a multivitamin after diagnosis had a 25% reduction in the risk of recurrence *. However, as discussed in the «Corrective Supplements» section , taking vitamins is only beneficial when there is a deficiency in the body.

Plant natural compounds targeting various signaling mechanisms may enhance the efficacy of classical chemotherapy drugs in the treatment of TNBC, and/or reduce the side effects of the latter.

Amygdalin increases in vitro (30 μM) apoptosis and reduces the adhesion of triple negative breast cancer cells (HS578t) *. At an amygdalin concentration of 80 mg/mL, cancer cell survival dropped to 15-30% (with an IC50≈ 50 mg/mL). Amygdalin (6-9 grams) is slowly (20-30 minutes) administered intravenously; oral administration of such a dose can lead to cyanide poisoning. However, the FDA is against the use of any form of amygdalin.

Curcumin and resveratrol * * are effective against TNBC in vitro and in vivoand can also reduce side effects *.

Epigallocatechin gallate (EGCG) inhibits uncontrolled cell growth and may delay the migration of cancer cells in TNBC *.

Carnosol blocks the cell cycle in the G2 phase in TNBC cells (MDA-MB-231) *.

• Other natural substances (luteolin; chalcone; piperine; quercetin; rutin; fisetin *; orange peel extract; rosemary extract; ursolic acid *) are effective in vitro, but this has not been clinically confirmed.

Obesity is an established risk factor for the progression and progression of triple-negative breast cancer *. Thus, in individuals with an elevated body mass index, a decrease in fat mass may contribute to the prevention and treatment of the disease. It should, however, be borne in mind that the «Weight Loss Program» † proposed in this work is experimental, not proven in clinical trials. To achieve the desired result for us with minimal side effects, you should turn to professionals.

Other means and methods.

• The combination of auranofin and vitamin C administered intraperitoneally to mice inhibited the growth of the grafted TNBC tumor (MDA-MB-231) by 12 times in 14 days of treatment compared to the control group *. The combination of these two available drugs may be effective not only against triple-negative breast cancer, but also other types of cancer by reducing inflammatory levels by suppressing the expression of PTGR1 (prostaglandin reductase 1). Although the mice tolerated the treatment well, the side effects of such treatment in humans are unknown. The dosage of vitamin C used in these experiments, in human terms, will be about 25 g/day; and the dosage of auranofin is 30 mg/day, which is 3-5 times higher than that usually prescribed orally in the treatment of rheumatoid arthritis.

• Combination treatment with paclitaxel and bazedoxifene has been shown in preclinical studies to act synergistically to inhibit viability, colony formation, and migration of TNBC cancer cells *. The dosage of bazedoxifene, determined in clinical studies, is 20-40 mg/day *.

At the same time, it was reported that some natural substances are able, on the contrary, to stimulate the growth of TNBC cells. For example, asparagine is a non-essential amino acid found in asparagus, many fruits, vegetables, meats and dairy products. Asparagine enhances protein and nucleotide synthesis by activating mTORC1, thereby promoting cell proliferation * * *.

Cancer cells can use sources of energy other than glucose, such as glutamine and fatty acids. Fatty acids may be the main energy source for TBNC cells *. The combination of inhibitors that block the metabolism of glutamine and fatty acids makes it possible to slow down the growth and migration of TNBC cells *.

As we can see, there are a large number of promising strategies for the fight against triple negative breast cancer. However, at the moment, almost all of them have no clinically proven benefit. It remains to be hoped that in the future they may be useful in the treatment of this most therapeutically severe case of breast cancer.

Other subtypes of breast cancer.

Some subtypes of cancer do not fit well into the accepted classification, or are a certain type of triple negative cancer. Although many of them do not respond to ER, HER2, AR, and other typical therapeutic receptors, other receptors may be involved in their treatment. Thus, most TNBC treatment items can be applied to them as well.

Quadruple negative breast cancer (QNBC) is a type of TNBC that does not express AR *. Such a name for this subtype has not yet entered clinical practice, and is preliminary. In contrast to AR-positive TNBC, which predominantly exhibits a luminal subtype, QNBC predominantly exhibits a basaloid subtype.

In the absence of others other than those used in TNBC, targets for QNBC treatment include ACSL4 (long chain acyl-CoA synthetase 4), SKP2 (S-phase kinase-associated protein 2), and EGFR (epidermal growth factor receptor).

ACSL4 is an enzyme that catalyzes the activation of long chain fatty acids. The expression of ACSL4 is inversely correlated with the expression of ER, PR, AR, HER2, EGFR and thus can serve as both a biomarker * and a target for QNBC therapy * *. Inhibition of tumor growth by inhibiting ACSL4 can be achieved with the antidiabetic drug rosiglitazone.

SKP2 is a subunit of the ubiquitin complex. SKP2 expression is significantly higher in tumors of patients with invasive breast cancer * and is inversely correlated with prognosis in invasive breast cancer. SKP2 can be bound by DHT and specific inhibitors of SKP2 are currently being developed.

Inflammatory breast cancer (IBC) is the most aggressive type of breast cancer and is characterized by rapid progression, resistance to chemotherapy, and early metastasis. The skin at the same time acquires the texture of «orange peel», and the breast itself swells, becomes painful and sensitive. IBC belongs to TNBC and is therefore insensitive to treatment with hormonal therapy and targeted HER2 therapy. Although this type accounts for only 2-5% of all breast cancers, it shows a poor prognosis with a 40% five-year survival compared to 87% for all breast cancers.

However, in IBC, in addition to standard therapy, one can try to use the therapeutic targets used in TNBC, with particular emphasis on anti-inflammatory, anti-angiogenic and anti-invasive therapy *.

Metformin inhibits the inflammatory pathway, and in addition, metformin-based combinatorial therapy is effective in xenografts involving inflammatory prostate and melanoma cell lines, whereas it is ineffective in noninflammatory cell lines from these lineages *.

• Hydrophilic statins, chronically taken by patients with an inflammatory subtype of cancer to lower cholesterol levels, can double the disease-free survival rate compared to patients not taking statins *.
Simvastatin further enhances the radiotherapy sensitivity of tumor-initiating TNBC cells and inflammatory subtype tumor cells, increasing the 3-year relapse-free survival. However, it may also show a protective effect against non-inflammatory cancer cells *.

Disulfiram forms a complex with copper and increases the intracellular concentration of copper without the help of membrane carriers. And copper, in turn, increases the oxidative stress caused by radiation and chemotherapy. Mice treated intraperitoneally with disulfiram (50 mg/kg) in combination with copper (0.5 mg/kg) had a three times lower growth rate of the grafted IBC tumor (SUM149) *.

Women with non-metastatic breast cancer who took disulfiram showed higher survival rates *. Although disulfiram may not be an effective monotherapy, it may help prolong the lives of patients with metastatic cancer *.

It has been reported that inflammatory breast cancer shows a higher frequency of viral sequences (71%) than sporadic breast cancer. Breast cancer cells in culture have been shown to contain beta-retroviral particles and secrete them *. This virus has been named Human breast tumor virus (HMTV). Unfortunately, no specific methods of combating this virus have been proposed.

In conclusion, although specific interventions may improve treatment outcomes, they do not replace general therapy.


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