Protein kinase C isoform expression as a predictor of disease outcome on endocrine therapy in breast cancer

Background

Although in vitro breast cancer models have demonstrated a role for protein kinase C (PKC) α and δ isoforms in endocrine insensitivity and resistance respectively, there is currently little clinical evidence to support these observations.

Aims

To define the pattern of PKC α and δ expression using breast cancer cell lines, with and without endocrine resistance, and also breast cancer samples, where expression can be correlated with clinicopathological and endocrine therapy outcome data.

Methods

PKC isoform expression was examined in tamoxifen responsive, oestrogen receptor positive (ER⁺), ER⁺ acquired tamoxifen resistant (TAM‐R) and oestrogen receptor negative (ER⁻) cell lines by western blotting and immunocytochemical analysis. PKC isoform expression was then examined by immunohistochemistry in archival breast cancer specimens from primary breast cancer patients with known clinical outcome in relation to endocrine response and survival on therapy.

Results

ER⁺ breast cancer cell lines expressed considerable PKC‐δ but barely detectable levels of PKC‐α, whereas ER⁻ cell lines expressed PKC‐α but little PKC‐δ. ER⁺ acquired TAM‐R cell lines expressed substantial levels of both PKC‐α and δ. In clinical samples, high PKC‐δ expression correlated to endocrine responsiveness whereas PKC‐α expression correlated to ER negativity. PKC‐δ was an independent predictor of duration of response to therapy. Patients showing a PKC‐δ⁺/PKC‐α⁻ phenotype had a six times longer endocrine response than patients with the PKC‐δ⁺/ PKC‐α⁺ phenotype (equating to tamoxifen resistance in vitro).

Conclusions

Levels of PKC‐α and δ expression appear to be indicative of response to anti‐oestrogen therapy and could be useful in predicting a patient''s suitability for endocrine therapy.Anti‐hormone therapies such as tamoxifen are widely used to treat breast cancer patients.¹ A small but significant number of patients receiving tamoxifen however will not respond or will develop resistance.^2,3 Many mechanisms have been suggested which may play a role in tamoxifen resistance but the mechanisms have not yet been fully elucidated.^4,5,6,7 Although rapid progress is being made in understanding the biology of oestrogen receptor (ER) function, the only predictive markers for endocrine therapy that currently yield sufficient levels of evidence to be recommended for routine practice, are ER and progesterone receptors, and to a lesser extent HER‐2 status.¹ Better ways of predicting which patients are suitable for endocrine therapy would prove useful in the fight against breast cancer. Expression of the signal transduction molecule, protein kinase C (PKC) is increased in breast cancer models of poor prognosis; for example, ER⁻ cell lines express significantly more PKC than ER⁺ cell lines.^8,9 However, multiple isoforms of PKC exist, with variation in their expression profile and mechanism of activation.^10,11,12 We have previously shown that ER⁺ MCF‐7 cell lines have high PKC‐δ and low PKC‐α expression, whereas ER⁻ MDA‐MB‐231 cells have high PKC‐α and low PKC‐δ expression. A wealth of literature now support these observations^13,14,15,16 linking PKC‐α expression to loss of ER expression and adverse cellular features,^13,15 and there is also emerging data that PKC‐δ expression can relate to loss of endocrine sensitivity in vitro.¹⁷ These studies did not however investigate the effect of PKC‐δ expression on clinical outcome. Moreover, a recent clinical study showed PKC‐α to be decreased in advanced breast cancer samples,¹⁸ suggesting that laboratory observations may not translate to the clinic.We have therefore established cell line models of tamoxifen resistance,^19,20 and used these models and well‐characterised clinical specimens^21,22 with known response to endocrine therapy, to study PKC expression. We have shown that PKC‐α and δ may prove useful in predicting whether patients will respond or not to endocrine therapy.