**3.1. Calcium oscillations as pharmacological targets in breast cancer**

An extensive clinical study including 431 patients with invasive ductal breast carcinoma and 46 patients with fibroadenoma analyzed the expression of anoctamin 1 (Ano1, TMEM16A), one of the members in the Ano family [103]. The study identified a correlation between Ano1 overexpression and the good prognosis in patients with lower clinical stage (stage I or II) of

Integrating these results, it is still premature to evaluate if Ano1 is or is not a predictive factor in good/poor prognosis in breast cancer patients, and if it is a suitable pharmacological target. It would be very useful to have more insights into the cellular mechanisms related to Ano1 activation. In our opinion, a possible scenario would be that the opening of calcium-activated chloride channels induces chloride efflux, membrane depolarization followed by the calcium influx through VGCCs. The high level of expression of calcium-activated chloride channels in breast cancer cells might be correlated with the tendency of these cells to be more depolarized with respect to the normal surrounding cells. However, this scenario would explain only the clinical data showing the association between Ano1 overexpression and the poor prognosis in

Muscarinic receptors have been described to be expressed in several non-neuronal cell types, including endothelial cells, smooth muscle cells, or bladder and gastrointestinal tract [104– 107]. In multiple malignancies, including breast, prostate, lung, ovary, pancreas, prostate, skin, stomach, uterus and colon cancer, muscarinic receptors have been demonstrated to con-

In particular, muscarinic receptors have also been described in normal murine mammary (NMuMG) cells, normal human-breast-derived MCF-10A cells, and homogenates of surgical samples derived from normal human mammary tissue by Western blot and radioligand bind-

detected in MCF-7 breast cancer cells [114]. Additionally, the expression of muscarinic acetylcholine receptors was evidenced in LM2, LM3, and LMM3 cell lines derived from spontaneous mammary adenocarcinomas developed in Balb/C mice [115–117]. These studies demonstrated the involvement of muscarinic receptors in the tumor cells proliferation, progression, and angiogenesis [115–117], by a mechanism involving the stimulation of the nitric oxide synthase

lipase C (PLC) and determine the release of calcium from intracellular reservoirs. In MCF7, human breast cancer cells evidenced the activation of MAP by muscarinic receptors [118]. Considering that acetylcholine exerts neurocrine, paracrine, and autocrine regulation of cancer cell proliferation, migration, etc., we might consider muscarinic receptors among the master-

and M5, are coupled with Gq/11 proteins that activate phospho-

and M4, was

ing assays [111–113]. The expression of muscarinic acetylcholine receptors, M<sup>3</sup>

the breast cancer or in patients with triple-negative breast cancers [103].

**2.8. Muscarinic acetylcholine receptors in breast cancer**

tribute to cell proliferation and cancer progression [108–110].

, M<sup>3</sup>

patients with breast cancer.

176 Calcium and Signal Transduction

activity [114].

Muscarinic receptors, M<sup>1</sup>

players in breast cancer.

Tetrodotoxin (TTX), a blocker of voltage-gated sodium channels (VGSCs), was demonstrated to diminish the number of oscillating MDA-MB-231 breast cancer cells and to reduce the amplitude and the frequency of the Ca2+ oscillations (i.e., spontaneous calcium transients) in the same cells [78]. While TTX had no effect on the basal calcium level in nonoscillating MDA-MB-231 breast cells, when applied in high extracellular K<sup>+</sup> conditions augmented the intracellular calcium concentration in both oscillating and nonoscillating MDA-MB-231 cells [78].

Previous studies have demonstrated that the blockade of VGSCs inhibits the invasion of endocrine-resistant breast cancer cells [119]. It is very interesting the correlation between the blockade of VGSCs and the reduction of spontaneous calcium oscillations. Therefore, considering that only metastatic cells are characterized by spontaneous calcium transients in comparison with low-metastatic or normal cells, a possible therapeutic strategy would be to diminish calcium transients by applying targeted pharmacological agents against VGSCs. An interesting approach would be to encapsulate voltage-gated sodium channels antagonists in charged liposomes and to target only highly metastatic breast cancer cells.
