Autophagy inhibition using hydrochloroquine in breast cancer patients: a pilot study

In response to various stresses, cells can launch a process of *self-eating*,
termed autophagy. Thereby, components of the cell are catabolically digested
via specific lysosomes called autophagosomes, to provide the cell with energy
and other necessary factors to serve as a temporary survival mechanism (Chen et
al. 2010).
Two major stressors that can be evaded by autophagy are important for cancer
progression and treatment sensitivity:
1. cells can respond with autophagy to cytotoxic treatment such as chemo- or
endocrine therapy, thereby leading to treatment insensitivity (Kondo et al.
2005; Chen et al. 2010), and
2. cells can survive severe hypoxia using autophagy (Rouschop et al. 2010), and
hypoxic cells themselves are refractory to chemo-, endocrine and radiotherapy.

Thus, tumor cells evade treatment induced cell death by launching a temporary
last survival mechanism. Inhibition of this pathway could lead to sensitization
for a variety of cancer treatment regimen, or to specific cell killing of tumor
associated hypoxic cells that would otherwise be refractory to radiotherapy.

Chloroquine (CQ), N*-(7-chloroquinolin-4-yl)-N,N-diethyl-pentane-1,4-diamine,
was discovered in 1934, and has widely been used as an effective and safe
anti-malarial and anti-rheumatoid agent since 1947. Later, CQ has been
rediscovered as a sensitizer of cytotoxic cancer therapies such as ionizing
radiation and chemotherapeutics, although the precise mechanism behind this has
remained largely unknown (Solomon and Lee 2009). Most recently, it was
discovered that CQ inhibits the process of autophagy by impairment of
autophagic vesicle clearance, as CQ accumulates in lysosomal vesicles. This has
now lead to several investigators proposing that CQ or one of its analogs can
be used to inhibit the autophagic pathway as an additive to other cytotoxic
treatments. Hydrochloroquine (HCQ, Plaquenil) is a CQ derivative with fewer
side effects than CQ, which has long been used as anti-malarial and
anti-rheumatoid agent. It can be safely used at high doses for extended periods
of time. Both CQ and HCQ are under investigation in clinical trials for
glioblastoma, small and non-small cell lung cancer, breast cancer, prostate
cancer, melanoma, renal cell carcinoma, and pancreatic cancer (for reviews see
Solomon and Lee 2009 and Chen et al. 2010). However, the effect of HCQ on tumor
tissue, autophagy and/or oxygenation has of yet not been studied in human
patients in vivo.

In this pilot study we intend to investigate the effect of HCQ on breast cancer
tissues. To this end, breast cancer patients that have given informed consent
for participation in the AFTER study (AMO 2010/312), but are not included as
their tissue biopsy is found to be ER/PgR negative, will be asked to take 800
mg once, and then 400 mg/day HCQ for 2 to 3 weeks until surgery. We will
compare tissue characteristics before and after treatment using HCQ, looking at
effects on markers for both hypoxia and autophagy using immunohistochemistry.
We expect that after treatment with HCQ tumor cells in hypoxic areas will no
longer be able to survive, thus decreasing the number of viable hypoxic cells
and increasing the amount of necrosis. This pilot study will serve as a proof
of principle for future studies into the effect of autophagy inhibition on
treatment sensitivity in breast cancer

Primary objective is to assess differences in endogenous hypoxia markers (CA9,
PAI-1, VEGF [Rademakers et al. 2008]) and autophagy (LC3b [Rouschop et al.
2010]) before and after short-term pre-surgical treatment with HCQ in breast
cancer patients.

secondary objectives will be:
- To compare post-treatment samples with matched (age, stage, histology)
samples of patient tissue in our breast tumor bank.
- To assess differences in putative crucial mediators in the autophagy pathway
currently under investigation, i.e. TRB3, ATF4, GRP78, LAMP3, etc.

If short-term treatment with HCQ leads to either less hypoxia/more necrosis in
tumors, or to signs of decreased autophagy, we intend to set up a larger trial
using HCQ as an additive to systemic or locoregional cytotoxic treatment.

In this pilot study we intend to investigate the effect of HCQ on breast cancer
tissues. To this end, breast cancer patients that have given informed consent
for participation in the AFTER study (AMO 2010/312), but are not included as
their tissue biopsy is found to be ER/PgR negative, will be asked to take 800
mg once, and then 400 mg/day HCQ for 2 to 3 weeks until surgery. We will
compare tissue characteristics before and after treatment using HCQ, looking at
effects on markers for both endogenous hypoxia markers (CA9, PAI-1, VEGF) and
autophagy (LC3b) before and after treatment with HCQ using standard
immunohistochemistry. This pilot study will serve as a proof of principle for
future studies into the effect of autophagy inhibition on treatment sensitivity
in breast cancer.

800 mg once, and then 400 mg/day HCQ for 2 to 3 weeks until surgery.

The expected side effects of HCQ during a short treatment period are slight.
The research takes place in the regular waiting period before surgery, so no
treatment delay occurs. This research can contribute significantly to knowledge
about the effect of HCQ on autophagy.